CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that “defatting” may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4–6 h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008 Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of l-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of l-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.     

Effect of fatty acids on physical properties of microsomes from isolated perfused rat liver

A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.     

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.     

MicroRNA-217 promotes ethanol-induced fat accumulation in hepatocytes by down-regulating SIRT1

Ethanol-mediated inhibition of hepatic sirtuin 1 (SIRT1) plays a crucial role in the pathogenesis of alcoholic fatty liver disease. Here, we investigated the underlying mechanisms of this inhibition by identifying a new hepatic target of ethanol action, microRNA-217 (miR-217). The role of miR-217 in the regulation of the effects of ethanol was investigated in cultured mouse AML-12 hepatocytes and in the livers of chronically ethanol-fed mice. In AML-12 hepatocytes and in mouse livers, chronic ethanol exposure drastically and specifically induced miR-217 levels and caused excess fat accumulation. Further studies revealed that overexpression of miR-217 in AML-12 cells promoted ethanol-mediated impairments of SIRT1 and SIRT1-regulated genes encoding lipogenic or fatty acid oxidation enzymes. More importantly, miR-217 impairs functions of lipin-1, a vital lipid regulator, in hepatocytes. Taken together, our novel findings suggest that miR-217 is a specific target of ethanol action in the liver and may present as a potential therapeutic target for treating human alcoholic fatty liver disease.     

Interleukin-38 alleviates hepatic steatosis through AMPK/autophagy-mediated suppression of endoplasmic reticulum stress in obesity models

Interleukin-38 (IL-38), recently recognized as a cytokine with anti-inflammatory properties that mitigate type 2 diabetes, has been associated with indicators of insulin resistance and nonalcoholic fatty liver disease (NAFLD). This study investigated the impact of IL-38 on hepatic lipid metabolism and endoplasmic reticulum (ER) stress. We assessed protein expression levels using Western blot analysis, while monodansylcadaverine staining was employed to detect autophagosomes in hepatocytes. Oil red O staining was utilized to examine lipid deposition. The study revealed elevated serum IL-38 levels in high-fat diet (HFD)-fed mice and IL-38 secretion from mouse keratinocytes. IL-38 treatment attenuated lipogenic lipid accumulation and ER stress markers in hepatocytes exposed to palmitate. Furthermore, IL-38 treatment increased AMP-activated protein kinase (AMPK) phosphorylation and autophagy. The effects of IL-38 on lipogenic lipid deposition and ER stress were nullified in cultured hepatocytes by suppressing AMPK through small interfering (si) RNA or 3-methyladenine (3MA). In animal studies, IL-38 administration mitigated hepatic steatosis by suppressing the expression of lipogenic proteins and ER stress markers while reversing AMPK phosphorylation and autophagy markers in the livers of HFD-fed mice. Additionally, AMPK siRNA, but not 3MA, mitigated IL-38-enhanced fatty acid oxidation in hepatocytes. In summary, IL-38 alleviates hepatic steatosis through AMPK/autophagy signaling-dependent attenuation of ER stress and enhancement of fatty acid oxidation via the AMPK pathway, suggesting a therapeutic strategy for treating NAFLD.     

Dantrolene prevents hepatic steatosis by reducing cytoplasmic Ca2+ level and ER stress

IntroductionOur previous studies demonstrated that dantrolene, a ryanodine receptor stabilizer, prevents endoplasmic reticulum (ER) stress in the heart. ER stress is a strong mediator of impaired lipid metabolism in the liver, thereby contributing to fatty liver disease. In this study, we investigated the effects of dantrolene on fatty liver disease in mice and ER stress in hepatocytes.Methods and resultsEight weeks old C57BL/6 mice were fed high-fat diet (HFD) for 8 weeks with or without the oral administration of dantrolene (100 mg/kg/day). The livers of mice without dantrolene (HFD group) showed severe fatty liver, whereas the livers of the mice treated with dantrolene (HFD + DAN group) only showed slightly fatty liver. To address the preventive effects of dantrolene, primary hepatocytes were cultured with palmitate in the presence or absence of dantrolene. Dantrolene reduced lipid load and prevents palmitate-induced increase in cytoplasmic Ca²⁺ and ER stress. Based on these findings, we propose that dantrolene is a potential new therapeutic agent against fatty liver disease.     

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Metabolic changes in lipids of rat plasma and hepatocytes induced by 17 alpha-ethynylestradiol treatment

Cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were used to investigate the change of lipid metabolism induced by administration of 17 alpha-ethynylestradiol. Treatment with 17 alpha-ethynylestradiol caused a decrease of rat plasma lipids (free cholesterol, cholesterol ester, triacylglycerol and phosphatidylcholine). No difference in the ability of urea nitrogen synthesis could be demonstrated between cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats and propylene glycol-treated rats (control). Total cholesterol and cholesterol ester contents of cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were increased in comparison with those of the control. Triacylglycerol content of cultured hepatocytes was not affected by 17 alpha-ethynylestradiol treatment. There was no difference in the composition of lipid content between liver tissues and cultured hepatocytes. These results suggest that hepatocytes isolated from livers maintain the character of livers treated with 17 alpha-ethynylestradiol or livers treated with propylene glycol. Free cholesterol and cholesterol ester synthesis from [14C]acetic acid by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were decreased to about 30% of the control. Triacylglycerol and polar lipid (phospholipid) synthesis from [14C]acetic acid were not affected by 17 alpha-ethynylestradiol treatment. Microsomal hydroxymethylglutaryl-CoA reductase activity of rat liver treated with 17 alpha-ethynylestradiol was decreased to about 50% of control. The secretions of free cholesterol, cholesterol ester, triacylglycerol, phosphatidylcholine, apolipoprotein BL and BS by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol treated rats were not decreased when compared with the control. Because lipid and apolipoprotein secretions from cultured hepatocytes treated with 17 alpha-ethynylestradiol were not decreased and cholesterol contents of liver tissues and cultured hepatocytes treated with 17 alpha-ethynylestradiol were increased and hepatic microsomal hydroxymethylglutaryl-CoA reductase activity was decreased by 17 alpha-ethynylestradiol treatment, it is suggested that the liver plays an important role in hypolipidemia induced by 17 alpha-ethynylestradiol by increasing the plasma lipid uptake mediated by an increased amount of lipoprotein receptors of liver membranes.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Influence of ethanol on the metabolism of perfused normal, fatty and cirrhotic rat livers

1. The influence of ethanol on the metabolism of perfused livers from normal rats and rats in various stages of development of dietary cirrhosis was studied. A choline-deficient, low-protein and high-fat diet was used. Results were obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in fatty and cirrhotic livers than in normal livers. Ethanol had no effect on the oxygen consumption of any of the various livers. After addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely in normal livers. Only a slight decrease in the carbon dioxide production occurred in fatty and cirrhotic livers. 3. With every type of liver glucose was released from the liver into the perfusion medium during the initial control period. This release continued after the addition of ethanol to the perfusion medium in experiments with normal and fatty livers, whereas with cirrhotic livers a marked uptake of glucose from the medium was found. A simultaneous release of the glycolytic end products lactate and pyruvate into the medium occurred. 4. The production of ketone bodies was equal in normal and early fatty livers (6 weeks on the fat diet). It was smaller in late fatty livers (3–4 months on the fatty diet) and in cirrhotic livers. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 11 to 67 with normal livers, from 12 to 16 with early fatty livers, from 13 to 26 with late fatty livers and from 21 to 55 with cirrhotic livers when the livers were perfused with a medium containing ethanol. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 1·2 to 8·4 in normal livers, from 2·0 to 2·8 in early fatty livers, from 1·2 to 2·4 in late fatty livers and from 2·1 to 4·0 in cirrhotic livers when ethanol was added to the medium. 6. The effects of ethanol on liver metabolism during the development of dietary cirrhosis are discussed and related to human fatty liver and cirrhosis during chronic ethanol consumption.     

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.     

Increase in portal flow induces c-myc expression in isolated perfused rat liver

We examined expression of the c-myc oncogene in isolated perfused livers to elucidate the mechanisms involved in triggering the proliferation of hepatocytes after partial hepatectomy (PH). During perfusion with a 1:1 mixture of Dulbecco's modified Eagle's medium and the oxygen transport fluid FC-43, rat livers were two-thirds resected (PH), and further perfused for 1 1/2 hours at the physiological portal flow throughout the perfusion. Expression of c-myc in the perfused livers with PH(+) was ten times higher than in those with PH(?). Furthermore, expression of c-myc in the PH(?) livers perfused with a threefold volume of the physiological portal flow was 5–10 times higher than that in the livers perfused with the physiological portal flow. The perfusates that passed through the livers did not induce DNA synthesis of primary cultured hepatocytes. These results suggest that an increase in the portal flow volume may act as a trigger for hepatocyte proliferation after PH. © 1993 Wiley-Liss, Inc.     

The control by vasopressin of carbohydrate and lipid metabolism in the perfused rat liver

Vasopressin-induced glucose release from the perfused livers of fed rats is diminished in the presence of insulin or following adrenal ablation. The reduced rate of glucose release following vasopressin treatment in the perfused livers of adrenalectomized rats was restored towards the control value by cortisol treatment in vivo.Vaspressin did not influence the total rate of fatty acid synthesis in the livers of fed rats perfused with medium containing glucose and two concentrations of lactate. The contribution of these precursors to hepatic fatty acid synthesis and CO₂ production was similarly uninfluenced by vasopressin.Vasopressin caused a transient increase in the release of K⁺ by the perfused liver which was observed within 2 min of hormone administration.These results are discussed in relation to the possible mode of action of vasopressin in the liver.     

Effects of ethynyloestradiol on the metabolism of [1-14C]-oleate by perfused livers and hepatocytes from female rats

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.     

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.     

The control by vasopressin of carbohydrate and lipid metabolism in the perfused rat liver.

Vasopressin-induced glucose release from the perfused livers of fed rats is diminished in the presence of insulin or following adrenal ablation. The reduced rate of glucose release following vasopressin treatment in the perfused livers of adrenalectomized rats was restored towards the control value by cortisol treatment in vivo. Vasopressin did not influence the total rate of fatty acid synthesis in the livers of fed rats perfused with medium containing glucose and two concentrations of lactate. The contribution of these precursors to hepatic fatty acid synthesis and CO2 production was similarly uninfluenced by vasopressin. Vasopressin casued a transient increase in the release of K+ by the perfused liver which was observed within 2 min of hormone administration. These results are discussed in relation to the possible mode of action of vasopressin in the liver.     

Fatty acid-binding protein expression in the liver: its regulation and relationship to the zonation of fatty acid metabolism

Summary Liver fatty acid-binding protein (L-FABP) is expressed in a declining gradient between the portal and central zones of the liver acinus. This paper discusses the results of experimental studies which address the questions: (a) What factors regulate L-FABP expression in liver and produce its acinar gradient? (b) What is the relationship between the acinar gradient of L-FABP and acinar gradients in the transport and metabolism of long-chain fatty acids? Both high-fat diets and clofibrate-treatment increase L-FABP proportionally at both extremes of the liver acinus and the small intestine, with preservation of the L-FABP gradient in both tissues. Female rats differ from males, however, in showing a greater hepatic abundance of L-FABP which is expressed almost equally throughout the acinus. Dietary studies show that L-FABP is induced with increased fatty acid flux derived from dietary fat but not from de novo hepatic fatty acid synthesis. Studies of the synthesis and utilization of fatty acids by hepatocytes isolated from the periportal and pericentral zones of the liver acinus suggest that the acinar gradient of L-FABP is not associated with differences in the instrinsic capacity of zone 1 and zone 3 hepatocytes to utilize or synthesize fatty acids. In addition, studies of the acinar uptake pattern of a fluorescent fatty acid derivative by isolated perfused livers indicate that the acinar distribution of L-FABP does not determine the pattern of fatty acid uptake in the intact acinus. Rather, the acinar gradient of L-FABP is most likely to represent a response to physiological conditions existing in the intact acinus which may include gradients in the flux of fatty acids, fatty acid metabolites and hormones.Abbreviations ALT Alanine Aminotransferase - FABP Fatty Acid Binding Protein - I-FABP Intestinal-type Fatty Acid Binding Protein - L-FABP Liver-type Fatty Acid Binding Protein - 12-NBD-stearate 12-(N-methyl)-N-(7-nitrobenzo-2-oxa-1, 3,-diazol-4-yl)amino)-octadecanoic acid     

Metabolism of chylomicrons by the isolated rat liver

Isolated livers perfused with washed corn oil chylomicrons labeled in vivo with palmitic acid-1-(14)C removed a large proportion of the chylomicrons. Slices from these livers oxidized chylomicron fatty acid esters to both carbon dioxide and acetoacetate. The liver slices also generated free fatty acids from chylomicron lipids and converted chylomicron triglycerides to phospholipids. Similar activities were observed in rat liver slices prepared shortly after the intravenous administration of chylomicrons to intact rats. The observed chylomicron uptake and lipid conversions were similar in livers from both fed and fasted rats. Fasting increased the oxidation of chylomicron fatty acid esters by livers labeled in vivo and by perfusion. In livers removed from intact rats given labeled chylomicrons, the triglyceride-(14)C to phospholipid-(14)C ratio was high, a finding unexpected if the liver had acquired this (14)C by removal of circulating fatty acids formed by extrahepatic lipolysis. These results demonstrate the ability of the liver to remove and utilize chylomicrons directly and suggest that direct removal accounts for a significant portion of the chylomicron fatty acids utilized by the liver of intact rats.     

Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.