Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria.

BACKGROUND: It is generally accepted that endothelial cells (ECs), primarily rely on glycolysis for ATP production, despite having functional mitochondria. However, it is also known that ECs are heterogeneous, and their phenotypic features depend on the vascular bed. Emerging evidence suggests that liver sinusoidal ECs (LSECs), located in the metabolically rich environment of the liver, show high metabolic plasticity. However, the substrate preference for energy metabolism in LSECs remains unclear. METHODS: Investigations were conducted in primary murine LSECs in vitro using the Seahorse XF technique for functional bioenergetic assays, untargeted mass spectrometry-based proteomics to analyse the LSEC proteome involved in energy metabolism pathways, liquid chromatography-tandem mass spectrometry-based analysis of acyl-carnitine species and Raman spectroscopy imaging to track intracellular palmitic acid. RESULTS: This study comprehensively characterized the energy metabolism of LSECs, which were found to depend on oxidative phosphorylation, efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids and glutamine. Furthermore, despite its high availability, palmitic acid was not directly oxidized in LSEC mitochondria, as evidenced by the acylcarnitine profile and etomoxir's lack of effect on oxygen consumption. However, together with L-carnitine, palmitic acid supported mitochondrial respiration, which is compatible with the chain-shortening role of peroxisomal ß-oxidation of long-chain fatty acids before further degradation and energy generation in mitochondria. CONCLUSIONS: LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment. The functional reliance of LSECs on oxidative phosphorylation, which is not a typical feature of ECs, remains to be determined.

Pomegranate Seed Oil as a Source of Conjugated Linolenic Acid (CLnA) Has No Effect on Atherosclerosis Development but Improves Lipid Profile and Affects the Expression of Lipid Metabolism Genes in apoE/LDLR-/- Mice.

The aim of this study was to evaluate the anti-atherosclerotic effect of pomegranate seed oil as a source of conjugated linolenic acid (CLnA) (cis-9,trans-11,cis-13; punicic acid) compared to linolenic acid (LnA) and conjugated linoleic acid (CLA) (cis-9,trans-11) in apoE/LDLR-/- mice. In the LONG experiment, 10-week old mice were fed for the 18 weeks. In the SHORT experiment, 18-week old mice were fed for the 10 weeks. Diets were supplied with seed oils equivalent to an amount of 0.5% of studied fatty acids. In the SHORT experiment, plasma TCh and LDL+VLDL cholesterol levels were significantly decreased in animals fed CLnA and CLA compared to the Control. The expression of PPARα in liver was four-fold increased in CLnA group in the SHORT experiment, and as a consequence the expression of its target gene ACO was three-fold increased, whereas the liver's expression of SREBP-1 and FAS were decreased in CLnA mice only in the LONG experiment. Punicic acid and CLA isomers were determined in the adipose tissue and liver in animals receiving pomegranate seed oil. In both experiments, there were no effects on the area of atherosclerotic plaque in aortic roots. However, in the SHORT experiment, the area of atherosclerosis in the entire aorta in the CLA group compared to CLnA and LnA was significantly decreased. In conclusion, CLnA improved the lipid profile and affected the lipid metabolism gene expression, but did not have the impact on the development of atherosclerotic plaque in apoE/LDLR-/- mice.

Actin-spectrin scaffold supports open fenestrae in liver sinusoidal endothelial cells.

Fenestrae are open transmembrane pores that are a structural hallmark of healthy liver sinusoidal endothelial cells (LSECs). Their key role is the transport of solutes and macromolecular complexes between the sinusoidal lumen and the space of Disse. To date, the biochemical nature of the cytoskeleton elements that surround the fenestrae and sieve plates in LSECs remain largely elusive. Herein, we took advantage of the latest developments in atomic force imaging and super-resolution fluorescence nanoscopy to define the organization of the supramolecular complex(es) that surround the fenestrae. Our data revealed that spectrin, together with actin, lines the inner cell membrane and provided direct structural support to the membrane-bound pores. We conclusively demonstrated that diamide and iodoacetic acid (IAA) affect fenestrae number by destabilizing the LSEC actin-spectrin scaffold. Furthermore, IAA induces rapid and repeatable switching between the open vs closed state of the fenestrae, indicating that the spectrin-actin complex could play an important role in controlling the pore number. Our results suggest that spectrin functions as a key regulator in the structural preservation of the fenestrae, and as such, it might serve as a molecular target for altering transendothelial permeability.

Tracking Fenestrae Dynamics in Live Murine Liver Sinusoidal Endothelial Cells.

The fenestrae of liver sinusoidal endothelial cells (LSECs) allow passive transport of solutes, macromolecules, and particulate material between the sinusoidal lumen and the liver parenchymal cells. Until recently, fenestrae and fenestrae-associated structures were mainly investigated using electron microscopy on chemically fixed LSECs. Hence, the knowledge about their dynamic properties has remained to date largely elusive. Recent progress in atomic force microscopy (AFM) has allowed the study of live cells in three dimensions (X, Y, and Z) over a prolonged time (t) and this at unprecedented speeds and resolving power. Hence, we employed the latest advances in AFM imaging on living LSECs. As a result, we were able to monitor the position, size, and number of fenestrae and sieve plates using four-dimensional AFM (X, Y, Z, and t) on intact LSECs in vitro. During these time-lapse experiments, dynamic data were collected on the origin and morphofunctional properties of the filtration apparatus of LSECs. We present structural evidence on single laying and grouped fenestrae, thereby elucidating their dynamic nature from formation to disappearance. We also collected data on the life span of fenestrae. More especially, the formation and closing of entire sieve plates were observed, and how the continuous rearrangement of sieve plates affects the structure of fenestrae within them was recorded. We observed also the dawn and rise of fenestrae-forming centers and defenestration centers in LSECs under different experimental conditions. Conclusion: Utilizing a multimodal biomedical high-resolution imaging technique we collected fine structural information on the life span, formation, and disappearance of LSEC fenestrae; by doing so, we also gathered evidence on three different pathways implemented in the loss of fenestrae that result in defenestrated LSECs.

Effect of conjugated linoleic acid and different type of dietary fat on serum lipid profile, liver enzymes activity and oxidative stress markers in Wistar rats

Background: Nutritional recommendations emphasize the need to limit consumption of saturated fatty acids and to increase the intake of polyunsaturated fatty acids in the prevention of non-communicable chronic diseases, particularly cardiovascular diseases. Among the fatty acids with health-related effects on the body, conjugated fatty acids are mentioned (i.e. CLA). Objective: The current study was designed to determine the effects of conjugated linoleic acid (CLA) on serum lipid profile, glucose, liver enzymes activity (AST and ALT), malonic dialdehyde (MDA) as well as lipid hydroperoxide (LPO) concentrations in rats fed diet differing in type of dietary fat. Material and methods: Male Wistar rats were divided into six groups and fed the following diets: control AIN-93G diet contained soybean oil (O) and diets with modification of fat source: butter (B) and margarine (M). The experimental diets were supplemented with 1% of conjugated linoleic acid (O+CLA, B+CLA, M+CLA). After 21 days the blood was collected and lipid profile, glucose, liver enzymes, MDA as well as LPO were analyzed. Results: The dietary treatments had no significant effect on the body weight and liver weight of the animals. The concentrations of total cholesterol (TC) and LDL+VLDL cholesterol were unchanged. Both experimental factors (fat source and CLA) had a significant influence on the TAG and HDL levels. Margarine (M) significantly increased the TAG concentration, whereas CLA had a significant impact on the TAG reduction (M+CLA). Glucose level was significantly decreased in all groups fed diets supplemented with CLA. Serum ALT significantly increased in all CLA groups. Fat source had statistically significant influence on the MDA concentration. The LPO level was significantly elevated in all CLA groups. There was statistically significant interaction of experimental factors (fat source and CLA supplementation) on LPO level. Conclusions: Margarine had an adverse effect on the rat's lipid profile. However, in the group fed with margarine, the addition of CLA decreased the concentration of TAG. Regardless of the type of the dietary fat, CLA supplementation increased the level of LPO in the blood serum of animals.

Rapid diagnostics of liver steatosis by Raman spectroscopy via fiber optic probe: a pilot study.

Raman spectroscopy via fiber optic probes omits some of the major limitations related to ex vivo preparation of tissue samples (e.g. fixation, freezing, cutting) and enables rapid registration of spectra, therefore, this technique has the potential to become a useful diagnostic tool. In this work, we evaluated the applicability of Raman spectroscopy via fiber optic probe for rapid assessment of lipid content in the liver in the context of its potential application as a tool to verify the degree of liver steatosis. Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder that is characterized by excessive lipid accumulation within hepatic tissue and is associated with insulin resistance and metabolic syndrome. Raman spectroscopy via fiber optic probe was applied to investigate the biochemical status of the liver in mild (mice fed a high-fat diet) and severe (db/db mice) models of NAFLD. A considerable increase in lipid content without substantial alterations in composition was observed in mild liver steatosis. In contrast, more severe liver steatosis caused not only a significant lipid content increase, but also hepatic cholesterol accumulation accompanied by significant loss of hepatic vitamin A content. Chemometric analysis based on average Raman spectra recorded via fiber optic probe provided discrimination of mild and severe liver steatosis and control livers with high sensitivity and specificity. In conclusion, our work demonstrates that a relatively simple Raman setup equipped with a commercial fiber optic probe combined with basic chemometric analysis enables rapid quantification of liver steatosis.

Changes induced by non-alcoholic fatty liver disease in liver sinusoidal endothelial cells and hepatocytes: spectroscopic imaging of single live cells at the subcellular level.

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent liver disorder worldwide, involving pathogenic mechanisms of liver sinusoidal endothelial cells (LSECs), hepatocytes and other liver cells. Here, we used a novel approach of label-free Raman confocal imaging to study primary LSECs and hepatocytes freshly isolated from the livers of mice with NAFLD induced by a high fat diet (HFD), in comparison to healthy controls. Our aim was to characterize changes in the biochemical composition in LSECs and hepatocytes that occur in a single cell at the subcellular level. LSECs from NAFLD livers displayed a significant increase in the intensity of marker bands of nuclear DNA that was not associated with changes in LSEC nucleus size. A number of changes in the cytoplasm of hepatocytes were identified. However, the most prominent change in hepatocytes was a substantial increase in the degree of unsaturation of LBs' (lipid bodies) lipids in NAFLD, suggesting an increase in the de novo lipogenesis of unsaturated lipids. The confocal Raman imaging of single live cells isolated from the liver provided a unique tool to better understand disease-induced cell-specific changes in the biochemical phenotype of primary liver cells.

Role of Mcpip1 in obesity-induced hepatic steatosis as determined by myeloid and liver-specific conditional knockouts.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1, alias Regnase 1) is a negative regulator of inflammation, acting through cleavage of transcripts coding for proinflammatory cytokines and by inhibition of NFκB activity. Moreover, it was demonstrated that MCPIP1 regulates lipid metabolism both in adipose tissue and in hepatocytes. In this study, we investigated the effects of tissue-specific Mcpip1 deletion on the regulation of hepatic metabolism and development of nonalcoholic fatty liver disease (NAFLD). We used control Mcpip1fl/fl mice and animals with deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/fl LysMCre ) and in hepatocytes (Mcpip1fl/fl AlbCre ), which were fed chow or a high-fat diet (HFD) for 12 weeks. Mcpip1fl/fl LysMCre mice fed a chow diet were characterized by a significantly reduced hepatic expression of genes regulating lipid and glucose metabolism, which subsequently resulted in low plasma glucose level and dyslipidemia. These animals also displayed systemic inflammation, demonstrated by increased concentrations of cytokines in the plasma and high Tnfa, Il6, IL1b mRNA levels in the liver and brown adipose tissue (BAT). Proinflammatory leukocyte infiltration into BAT, together with low expression of Ucp1 and Ppargc1a, resulted in hypothermia of 22-week-old Mcpip1fl/fl LysMCre mice. On the other hand, there were no significant changes in phenotype in Mcpip1fl/fl AlbCre mice. Although we detected a reduced hepatic expression of genes regulating glucose metabolism and ß-oxidation in these mice, they remained asymptomatic. Upon feeding with a HFD, Mcpip1fl/fl LysMCre mice did not develop obesity, glucose intolerance, nor hepatic steatosis, but were characterized by low plasma glucose level and dyslipidemia, along with proinflammatory phenotype. Mcpip1fl/fl AlbCre animals, following a HFD, became hypercholesterolemic, but accumulated lipids in the liver at the same level as Mcpip1fl/fl mice, and no changes in the level of soluble factors tested in the plasma were detected. We have demonstrated that Mcpip1 protein plays an important role in the liver homeostasis. Depletion of Mcpip1 in myeloid leukocytes, followed by systemic inflammation, has a more pronounced effect on controlling liver metabolism and homeostasis than the depletion of Mcpip1 in hepatocytes.

Differential response of liver sinusoidal endothelial cells and hepatocytes to oleic and palmitic acid revealed by Raman and CARS imaging.

Excess circulating fatty acids contribute to endothelial dysfunction that subsequently aggravates the metabolic conditions such as fatty liver diseases. However, the exact mechanism of this event is not fully understood, and the investigation on the effect of a direct exposure to fatty acids together with their subsequent fate is of interest. In this work we employed a chemically specific and label-free techniques such as Raman and CARS microscopies, to investigate the process of lipid droplets (LDs) formation in endothelial cells and hepatocytes after exposure to oleic and palmitic acid. We aimed to observe the changes in the composition of LDs associated with metabolism and degradation of lipids. We were able to characterize the diversity in the formation of LDs in endothelium as compared to hepatocytes, as well as the differences in the formation of LDs and degradation manner with respect to the used fatty acid. Thus, for the first time the spectral characteristics of LDs formed in endothelial cells after incubation with oleic and palmitic acid is presented, including the time-dependent changes in their chemical composition.

In Vivo Magnetic Resonance Imaging-Based Detection of Heterogeneous Endothelial Response in Thoracic and Abdominal Aorta to Short-Term High-Fat Diet Ascribed to Differences in Perivascular Adipose Tissue in Mice.

Background Long-term feeding with a high-fat diet (HFD) induces endothelial dysfunction in mice, but early HFD-induced effects on endothelium have not been well characterized. Methods and Results Using an magnetic resonance imaging-based methodology that allows characterization of endothelial function in vivo, we demonstrated that short-term (2 weeks) feeding with a HFD to C57BL/6 mice or to E3L.CETP mice resulted in the impairment of acetylcholine-induced response in the abdominal aorta (AA), whereas, in the thoracic aorta (TA), the acetylcholine-induced response was largely preserved. Similarly, HFD resulted in arterial stiffness in the AA, but not in the TA. The difference in HFD-induced response was ascribed to distinct characteristics of perivascular adipose tissue in the TA and AA, related to brown- and white-like adipose tissue, respectively, as assessed by histology, immunohistochemistry, and Raman spectroscopy. In contrast, short-term HFD-induced endothelial dysfunction could not be linked to systemic insulin resistance, changes in plasma concentration of nitrite, or concentration of biomarkers of glycocalyx disruption (syndecan-1 and endocan), endothelial inflammation (soluble form of vascular cell adhesion molecule 1, soluble form of intercellular adhesion molecule 1 and soluble form of E-selectin), endothelial permeability (soluble form of fms-like tyrosine kinase 1 and angiopoietin 2), and hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). Conclusions Short-term feeding with a HFD induces endothelial dysfunction in the AA but not in the TA, which could be ascribed to a differential response of perivascular adipose tissue to a HFD in the AA versus TA. Importantly, early endothelial dysfunction in the AA is not linked to elevation of classical systemic biomarkers of endothelial dysfunction.