Over-Expression of Intestinal Alkaline Phosphatase Attenuates Atherosclerosis.

[Figure: see text].

Neutrophil Elastase Triggers the Release of Macrophage Extracellular Traps: Relevance to Cystic Fibrosis.

Neutrophil extracellular traps increase cystic fibrosis (CF) airway inflammation. We hypothesized that macrophage exposure to neutrophil elastase (NE) would trigger the release of macrophage extracellular traps (METs), a novel mechanism to augment NE-induced airway inflammation in CF. Experiments were performed using human blood monocyte derived macrophages (hBMDM) from patients with and without CF to test specific mechanisms associated with MET release, and MET release by NE was confirmed in alveolar macrophages from Cftr-null and wild-type littermate mice exposed to intratracheal NE in vivo. Human BMDM were exposed to FITC-NE, and intracellular FITC-NE was localized to cytoplasmic and nuclear domains. Intracellular NE was proteolytically active as indicated by DQ-Elastin substrate cleavage. NE (100 to 500 nM) significantly increased extracellular PicoGreen fluorescence consistent with DNA release/ MET release from hBMDM in the absence of cell death. MET release was further confirmed by confocal microscopy in hBMDM treated with NE, and in alveolar macrophages from Cftr-null and wild-type littermate mice that had been exposed to intratracheal NE. NE-triggered MET release was associated with H3 citrullination detected by immunofluorescence assays and with partial cleavage of histone H3 but not H4. Exposure to NE caused release of METs from both CF and non-CF hBMDM in vitro and murine alveolar macrophages in vivo. MET release was associated with NE-activated H3 clipping, a mechanism associated with chromatin decondensation, a prerequisite for METs.

Neutrophil elastase-regulated macrophage sheddome/secretome and phagocytic failure.

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.

Dietary Supplementation with Galactooligosaccharides Attenuates High-Fat, High-Cholesterol Diet-Induced Glucose Intolerance and Disruption of Colonic Mucin Layer in C57BL/6 Mice and Reduces Atherosclerosis in Ldlr-/- Mice.

BACKGROUND: A Western-type diet (WD), rich in fat and cholesterol but deficient in fiber, induces development of diabetes and atherosclerosis. Colonic bacteria use the gut's mucous lining as an alternate energy source during periods of fiber deficiency, resulting in intestinal barrier erosion. OBJECTIVE: We hypothesized that supplementing a WD with galactooligosaccharide (GOS) fiber would attenuate WD-induced mucin layer disruption and attenuate development of metabolic diseases. METHODS: C57BL/6 mice (both sexes, 8-10 wk of age) were fed a standard rodent diet (TD7012, reference) or a high-fat, high-cholesterol-containing WD (TD88137, 21% fat, 0.15% cholesterol, 19.5% caesin) or a WD supplemented with 5% GOS fiber (TD170432, WD + GOS) for 16 wk. WD-fed mice that were gavaged daily with curcumin (100 mg/kg) served as positive controls. Glucose tolerance, colonic mucin layer, gene expression, and circulating macrophage/neutrophil levels were determined. Hyperlipidemic Ldlr-/- mice (both sexes, 8-10 wk of age) fed a WD with or without GOS supplementation (for 16 wk) were used to assess plasma LPS and atherosclerosis. Effects of dietary supplementation on different parameters were compared for each genotype. RESULTS: Compared with a WD, glucose tolerance was significantly improved in male C57BL/6 mice fed a WD + GOS (mean ± SEM: AUC = 53.6 ± 43.9 compared with 45.4 ± 33.3 g ⋅ min/dL; P = 0.015). Continuity of colonic mucin layer (MUC-2 expression) was improved in mice receiving GOS supplementation, indicating improved intestinal barrier. GOS supplementation also reduced circulating macrophages (30% decrease) and neutrophils (60% decrease), suggesting diminished systemic inflammation. In Ldlr-/- mice, GOS supplementation significantly reduced plasma LPS concentrations (mean ± SEM: 0.81 ±  0.43 EU/mL compared with 0.32 ± 0.26 EU/mL, P   < 0.0001, in females and 0.56 ± 0.24 EU/mL compared with 0.34 ± 0.12 EU/mL, P = 0.036, in males), improved glucose tolerance in male mice, and attenuated atherosclerotic lesion area (mean ± SEM: 54.2% ± 6.19% compared with 43.0% ± 35.12%, P   = 0.0006, in females and 54.6% ± 3.99% compared with 43.1% ± 8.11%, P = 0.003, in males). CONCLUSIONS: GOS fiber supplementation improves intestinal barrier in C57BL/6 and Ldlr-/- mice and significantly attenuates WD-induced metabolic diseases and, therefore, may represent a novel strategy for management of these diseases.

Identifying important parameters in the inflammatory process with a mathematical model of immune cell influx and macrophage polarization.

In an inflammatory setting, macrophages can be polarized to an inflammatory M1 phenotype or to an anti-inflammatory M2 phenotype, as well as existing on a spectrum between these two extremes. Dysfunction of this phenotypic switch can result in a population imbalance that leads to chronic wounds or disease due to unresolved inflammation. Therapeutic interventions that target macrophages have therefore been proposed and implemented in diseases that feature chronic inflammation such as diabetes mellitus and atherosclerosis. We have developed a model for the sequential influx of immune cells in the peritoneal cavity in response to a bacterial stimulus that includes macrophage polarization, with the simplifying assumption that macrophages can be classified as M1 or M2. With this model, we were able to reproduce the expected timing of sequential influx of immune cells and mediators in a general inflammatory setting. We then fit this model to in vivo experimental data obtained from a mouse peritonitis model of inflammation, which is widely used to evaluate endogenous processes in response to an inflammatory stimulus. Model robustness is explored with local structural and practical identifiability of the proposed model a posteriori. Additionally, we perform sensitivity analysis that identifies the population of apoptotic neutrophils as a key driver of the inflammatory process. Finally, we simulate a selection of proposed therapies including points of intervention in the case of delayed neutrophil apoptosis, which our model predicts will result in a sustained inflammatory response. Our model can therefore provide hypothesis testing for therapeutic interventions that target macrophage phenotype and predict outcomes to be validated by subsequent experimentation.

Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice.

Intracellular cholesterol transport proteins move cholesterol to different subcellular compartments and thereby regulate its final metabolic fate. In hepatocytes, for example, delivery of high-density lipoprotein (HDL)-associated cholesterol for bile acid synthesis or secretion into bile facilitates cholesterol elimination from the body (anti-atherogenic effect), whereas delivery for esterification and subsequent incorporation into apolipoprotein B-containing atherogenic lipoproteins (e.g. very-low-density lipoprotein (VLDL)) enhances cholesterol secretion into the systemic circulation (pro-atherogenic effect). Intracellular cholesterol transport proteins such as sterol carrier protein-2 (SCP2) should, therefore, play a role in regulating these pro- or anti-atherosclerotic processes. Here, we sought to evaluate the effects of SCP2 deficiency on the development of diet-induced atherosclerosis. We generated LDLR-/- mice deficient in SCP2/SCPx (LS) and examined the effects of this deficiency on Western diet-induced atherosclerosis. SCP2/SCPx deficiency attenuated atherosclerosis in LS mice by >80% and significantly reduced plasma cholesterol and triglyceride levels. Investigation of the likely underlying mechanisms revealed a significant reduction in intestinal cholesterol absorption (given as an oral gavage) in SCP2/SCPx-deficient mice. Consistently, siRNA-mediated knockdown of SCP2 in intestinal cells significantly reduced cholesterol uptake. Furthermore, hepatic triglyceride/VLDL secretion from the liver or hepatocytes isolated from SCP2/SCPx-deficient mice was significantly reduced. These results indicate an important regulatory role for SCP2 deficiency in attenuating diet-induced atherosclerosis by limiting intestinal cholesterol absorption and decreasing hepatic triglyceride/VLDL secretion. These findings suggest targeted inhibition of SCP2 as a potential therapeutic strategy to reduce Western diet-induced dyslipidemia and atherosclerosis.

Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression.

BACKGROUND: The associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function. METHODS: Renal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls. RESULTS: The Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia. CONCLUSIONS: Butyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.

Leutusome: A Biomimetic Nanoplatform Integrating Plasma Membrane Components of Leukocytes and Tumor Cells for Remarkably Enhanced Solid Tumor Homing.

Cell membrane-camouflaged nanoparticles have appeared as a promising platform to develop active tumor targeting nanomedicines. To evade the immune surveillance, we designed a composite cell membrane-camouflaged biomimetic nanoplatform, namely, leutusome, which is made of liposomal nanoparticles incorporating plasma membrane components derived from both leukocytes (murine J774A.1 cells) and tumor cells (head and neck tumor cells HN12). Exogenous phospholipids were used as building blocks to fuse with two cell membranes to form liposomal nanoparticles. Liposomal nanoparticles made of exogenous phospholipids only or in combination with one type of cell membrane were fabricated and compared. The anticancer drug paclitaxel (PTX) was used to make drug-encapsulating liposomal nanoparticles. Leutusome resembling characteristic plasma membrane components of the two cell membranes were examined and confirmed in vitro. A xenograft mouse model of head and neck cancer was used to profile the blood clearance kinetics, biodistribution, and antitumor efficacy of the different liposomal nanoparticles. The results demonstrated that leutusome obtained prolonged blood circulation and was most efficient accumulating at the tumor site (79.1 ± 6.6% ID per gram of tumor). Similarly, leutusome composed of membrane fractions of B16 melanoma cells and leukocytes (J774A.1) showed prominent accumulation within the B16 tumor, suggesting the generalization of the approach. Furthermore, PTX-encapsulating leutusome was found to most potently inhibit tumor growth while not causing systemic adverse effects.

A novel role of astrocyte elevated gene-1 (AEG-1) in regulating nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease in the Western world. However, an optimum therapy for NASH is yet to be established, mandating more in-depth investigation into the molecular pathogenesis of NASH to identify novel regulatory molecules and develop targeted therapies. Here, we unravel a unique function of astrocyte elevated gene-1(AEG-1)/metadherin in NASH using a transgenic mouse with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) and a conditional hepatocyte-specific AEG-1 knockout mouse (AEG-1ΔHEP ). Alb/AEG-1 mice developed spontaneous NASH whereas AEG-1ΔHEP mice were protected from high-fat diet (HFD)-induced NASH. Intriguingly, AEG-1 overexpression was observed in livers of NASH patients and wild-type (WT) mice that developed steatosis upon feeding HFD. In-depth molecular analysis unraveled that inhibition of peroxisome proliferator-activated receptor alpha activity resulting in decreased fatty acid ß-oxidation, augmentation of translation of fatty acid synthase resulting in de novo lipogenesis, and increased nuclear factor kappa B-mediated inflammation act in concert to mediate AEG-1-induced NASH. Therapeutically, hepatocyte-specific nanoparticle-delivered AEG-1 small interfering RNA provided marked protection from HFD-induced NASH in WT mice. CONCLUSION: AEG-1 might be a key molecule regulating initiation and progression of NASH. AEG-1 inhibitory strategies might be developed as a potential therapeutic intervention in NASH patients. (Hepatology 2017;66:466-480).

Curcumin improves intestinal barrier function: modulation of intracellular signaling, and organization of tight junctions.

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as type 2 diabetes and atherosclerosis) has shifted the focus from high-fat high-cholesterol containing Western-type diet (WD)-induced changes in gut microbiota per se to release of gut bacteria-derived products (e.g., LPS) into circulation due to intestinal barrier dysfunction as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. We demonstrated earlier that oral supplementation with curcumin attenuates WD-induced development of type 2 diabetes and atherosclerosis. Poor bioavailability of curcumin has precluded the establishment of a causal relationship between oral supplementation and it is in vivo effects. We hypothesized that curcumin attenuates WD-induced chronic inflammation and associated metabolic diseases by modulating the function of intestinal epithelial cells (IECs) and the intestinal barrier function. The objective of the present study was to delineate the underlying mechanisms. The human IEC lines Caco-2 and HT-29 were used for these studies and modulation of direct as well as indirect effects of LPS on intracellular signaling as well as tight junctions were examined. Pretreatment with curcumin significantly attenuated LPS-induced secretion of master cytokine IL-1ß from IECs and macrophages. Furthermore, curcumin also reduced IL-1ß-induced activation of p38 MAPK in IECs and subsequent increase in expression of myosin light chain kinase involved in the phosphorylation of tight junction proteins and ensuing disruption of their normal arrangement. The major site of action of curcumin is, therefore, likely the IECs and the intestinal barrier, and by reducing intestinal barrier dysfunction, curcumin modulates chronic inflammatory diseases despite poor bioavailability.

Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile.

While HDL-associated unesterified or free cholesterol (FC) is thought to be rapidly secreted into the bile, the fate of HDL-associated cholesteryl esters (HDL-CEs) that represent >80% of HDL-cholesterol, is only beginning to be understood. In the present study, we examined the hypothesis that intracellular cholesterol transport proteins [sterol carrier protein 2 (SCP2) and fatty acid binding protein-1 (FABP1)] not only facilitate CE hydrolase-mediated hydrolysis of HDL-CEs, but also enhance elimination of cholesterol into bile. Adenovirus-mediated overexpression of FABP1 or SCP2 in primary hepatocytes significantly increased hydrolysis of HDL-[(3)H]CE, reduced resecretion of HDL-CE-derived FC as nascent HDL, and increased its secretion as bile acids. Consistently, the flux of [(3)H]cholesterol from HDL-[(3)H]CE to biliary bile acids was increased by overexpression of SCP2 or FABP1 in vivo and reduced in SCP2(-/-) mice. Increased flux of HDL-[(3)H]CE to biliary FC was noted with FABP1 overexpression and in SCP2(-/-) mice that have increased FABP1 expression. Lack of a significant decrease in the flux of HDL-[(3)H]CE to biliary FC or bile acids in FABP1(-/-) mice indicates the likely compensation of its function by an as yet unidentified mechanism. Taken together, these studies demonstrate that FABP1 and SCP2 facilitate the preferential movement of HDL-CEs to bile for final elimination.

Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis.

Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.

Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr-/- mice.

The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR(-/-) background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.

Liver-specific cholesteryl ester hydrolase deficiency attenuates sterol elimination in the feces and increases atherosclerosis in ldlr-/- mice.

OBJECTIVE: Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. APPROACH AND RESULTS: Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in low density lipoprotein receptor knock out(-/-) mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out(-/-)Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [(3)H]-CE-labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. CONCLUSIONS: Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.

Curcumin and chronic kidney disease (CKD): major mode of action through stimulating endogenous intestinal alkaline phosphatase.

Curcumin, an active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa), has significant anti-inflammatory properties. Chronic kidney disease (CKD), an inflammatory disease, can lead to end stage renal disease resulting in dialysis and transplant. Furthermore, it is frequently associated with other inflammatory disease such as diabetes and cardiovascular disorders. This review will focus on the clinically relevant inflammatory molecules that play a role in CKD and associated diseases. Various enzymes, transcription factors, growth factors modulate production and action of inflammatory molecules; curcumin can blunt the generation and action of these inflammatory molecules and ameliorate CKD as well as associated inflammatory disorders. Recent studies have shown that increased intestinal permeability results in the leakage of pro-inflammatory molecules (cytokines and lipopolysaccharides) from gut into the circulation in diseases such as CKD, diabetes and atherosclerosis. This change in intestinal permeability is due to decreased expression of tight junction proteins and intestinal alkaline phosphatase (IAP). Curcumin increases the expression of IAP and tight junction proteins and corrects gut permeability. This action reduces the levels of circulatory inflammatory biomolecules. This effect of curcumin on intestine can explain why, despite poor bioavailability, curcumin has potential anti-inflammatory effects in vivo and beneficial effects on CKD.

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE.

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing.

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice.

Cellular cholesterol homeostasis is increasingly being recognized as an important determinant of the inflammatory status of macrophages, and a decrease in cellular cholesterol levels polarizes macrophages toward an anti-inflammatory or M2 phenotype. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of stored intracellular cholesteryl esters (CE) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have reported earlier reduced atherosclerosis as well as lesion necrosis and improved insulin sensitivity (due to decreased adipose tissue inflammation) in macrophage-specific CEH transgenic (CEHTg) mice in the LDLR(-/-) background. In the present study, we examined the effects of reduced intracellular accumulation of CE in CEHTg macrophages in an established diabetic mouse model, namely the leptin-deficient ob/ob mouse. Macrophage-specific transgenic expression of CEH improved glucose tolerance in ob/ob-CEHTg mice significantly compared with ob/ob nontransgenic littermates, but with no apparent change in macrophage infiltration into the adipose tissue. However, there was a significant decrease in hepatic lipid accumulation in ob/ob-CEHTg mice. Consistently, decreased [(14)C]acetate incorporation into total lipids and triglycerides was noted in precision-cut liver slices from ob/ob-CEHTg mice. In the primary hepatocyte-macrophage coculture system, macrophages from CEHTg mice significantly reduced the incorporation of [(14)C]acetate into triglycerides in hepatocytes, indicating a direct effect of macrophages on hepatocyte triglyceride biosynthesis. Kupffer cells isolated from ob/ob-CEHTg mice were polarized toward an anti-inflammatory M2 (Ly6C(lo)) phenotype. Taken together, these studies demonstrate that transgenic overexpression of CEH in macrophages polarizes hepatic macrophages (Kupffer cells) to an anti-inflammatory M2 phenotype that attenuates hepatic lipid synthesis and accumulation.

Measurement of In Vivo VLDL and Chylomicron Secretion.

Intestinal lipid absorption as well as secretion of absorbed lipids as chylomicrons by the enterocytes is a direct measure of the availability of dietary lipids. Measurement of this parameter is central to the understanding of the influence of diet on plasma lipids, specifically when modulation of intestinal lipid absorption by targeted interventions is being examined. In the post-prandial state, very low-density lipoprotein (VLDL) secreted from the liver represent the major source of plasma lipids and rate of VLDL secretion reports on hepatic lipid homeostasis. Here, we describe the methods to specifically measure secretion of chylomicron and VLDL in vivo. Tight regulation of dietary lipid absorption (chylomicron secretion) and hepatic secretion of VLDL underlies the development of dyslipidemia preceding hepatic lipid accumulation seen in non-alcoholic fatty liver disease (NAFLD) and subsequent progression to non-alcoholic steatohepatitis (NASH) underscoring the importance of measurement of lipoprotein secretion in vivo.