Hepatocyte [H-3]-palmitate uptake: effect of albumin surface charge modification

The role of plasma proteins on the cellular uptake of lipophilic substrates has perplexed investigators for many years. We tested the hypothesis that an ionic interaction between the protein-ligand complex and hepatocyte surface may be responsible for supplying more ligand to the cell for uptake. The surface-charged groups on albumin were modified to yield proteins having a range of isoelectric points (ALB, ALBs, ALBm, ALBe had values of 4.8-5.0, 4.5-4.7, 3.0-3.5, 8.4-8.6, respectively). [H-3]-Palmitate uptake studies were performed with adult rat hepatocyte suspensions using similar unbound ligand fractions in the presence of the different binding proteins. Mass spectrometry, isoelectric focusing (pI), and heptane : water partitioning were used to determine protein molecular weight, pI, and protein-palmitate equilibrium binding constant, respectively. Hepatocyte [H-3]-palmitate clearance in the presence of ALBs and ALBm were significantly lower (p < 0.05) than ALB, whereas [H-3]-palmitate clearance in the presence of ALBe was significantly higher (p < 0.05) than ALB. The data were consistent with the notion that ionic interactions between extracellular protein-ligand complexes and the hepatocyte surface facilitate the uptake of long-chain fatty acids.

Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [H-3] palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [H-3] palmitate and metabolites were measured in four experimentalgroups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [H-3] palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [H-3] palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [H-3] palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.

Reduced hepatic extraction of palmitate in steatosis correlated to lower level of liver fatty acid binding protein

Nonalcoholic fatty liver disease is the most common of all liver diseases. The hepatic disposition [H-3]palmitate and its low-molecular-weight metabolites in perfused normal and steatotic rat liver were studied using the multiple indicator dilution technique and a physiologically based slow diffusion/bound pharmacokinetic model. The steatotic rat model was established by administration of 17alpha-ethynylestradiol to female Wistar rats. Serum biochemistry markers and histology of treated and normal animals were assessed and indicated the presence of steatosis in the treatment group. The steatotic group showed a significantly higher alanine aminotransferase-to-aspartate aminotransferase ratio, lower levels of liver fatty acid binding protein and cytochrome P-450, as well as microvesicular steatosis with an enlargement of sinusoidal space. Hepatic extraction for unchanged [H-3]palmitate and production of low-molecular-weight metabolites were found to be significantly decreased in steatotic animals. Pharmacokinetic analysis suggested that the reduced extraction and sequestration for palmitate and its metabolites was mainly attributed to a reduction in liver fatty acid binding protein in steatosis.

Expression analysis of a human hepatic cell line in response to palmitate

Saturated fat plays a role in common debilitating diseases such as obesity, type 2 diabetes, and coronary heart disease. It is also clear that certain fatty acids act as regulators of metabolism via both direct and indirect signalling of target tissues. As the molecular mechanisms of saturated fatty acid signalling in the liver are poorly defined, hepatic gene expression analysis was undertaken in a human hepatocyte cell line after incubation with palmitate. Profiling of mRNA expression using cDNA microarray analysis revealed that 162 of approximately 18,000 genes tested were differentially expressed after incubation with palmitate for 48 h. Altered transcription profiles were observed in a wide variety of genes, including genes involved in lipid and cholesterol transport, cholesterol catabolism, cell growth and proliferation, cell signalling, P-oxidation, and oxidative stress response. While palinitate signalling has been examined in pancreatic beta-cells, this is the first report showing that palmitate regulates expression of numerous genes via direct molecular signalling mechanisms in liver cells. (C) 2005 Elsevier Inc. All rights reserved.

Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [H-3]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (-14 +/- 1 mV, n = 12, mean +/- S.E., substituting choline for Na+) or hyperpolarization (-46 +/- 3 mV, n = 12, mean +/- S.E., substituting nitrate for Cl-). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate-albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [H-3]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [H-3]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl- ion. This work contrasts with that for the extraction of [H-3]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [H-3]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [H-3]-palmitate extraction fraction in the IPL.

A compartmental model of hepatic disposition kinetics: 1. Model development and application to linear kinetics

The conventional convection-dispersion model is widely used to interrelate hepatic availability (F) and clearance (Cl) with the morphology and physiology of the liver and to predict effects such as changes in liver blood flow on F and Cl. The extension of this model to include nonlinear kinetics and zonal heterogeneity of the liver is not straightforward and requires numerical solution of partial differential equation, which is not available in standard nonlinear regression analysis software. In this paper, we describe an alternative compartmental model representation of hepatic disposition (including elimination). The model allows the use of standard software for data analysis and accurately describes the outflow concentration-time profile for a vascular marker after bolus injection into the liver. In an evaluation of a number of different compartmental models, the most accurate model required eight vascular compartments, two of them with back mixing. In addition, the model includes two adjacent secondary vascular compartments to describe the tail section of the concentration-time profile for a reference marker. The model has the added flexibility of being easy to modify to model various enzyme distributions and nonlinear elimination. Model predictions of F, MTT, CV2, and concentration-time profile as well as parameter estimates for experimental data of an eliminated solute (palmitate) are comparable to those for the extended convection-dispersion model.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Effect of fatty acids, glucose, and insulin on hepatic glucose uptake and glycolysis

Objective:. There is evidence from in vitro studies that fatty acids can inhibit glucose uptake in liver. However, it is uncertain whether this happens in vivo when the liver is exposed to high levels of glucose and insulin, in combination with fatty acids, after a mixed meal. This study determined the effects of a combination of fatty acids and insulin on glucokinase (GK) activity and glycolysis in primary rat hepatocytes. Methods: Hepatocytes were cultured with 15 mM glucose and 2 or 10 nM insulin in combination with the fatty acids palmitate, oleate, linoleate, eicosapentaenoic acid, or docosahexaenoic acid. Total GK activity and the proportion of GK in the,active, unbound state were measured to determine the effect of fatty acid on the activity and cellular localization of GK. Glucose phosphorylation and glycolysis were measured in intact cells. Lactate and pyruvate synthesis and the accumulation of ketone bodies were also estimated. Results: Palmitate and eicosapentaenoic acid lowered total GK activity in the presence of 2 nM insulin, but not with 10 nM insulin. In contrast, oleate, linoleate, and docosahexaenoic acid did not alter GK activity. None of the fatty acids tested inhibited glucose phosphorylation or glycolysis in intact rat hepatocytes. In addition, GK activity was unaffected by insulin concentration. Conclusion: Some fatty acids can act to inhibit GK activity in primary hepatocytes. However, there was no,evidence that this decrease in GK activity impaired glucose phosphorylation or glycolysis. Glucose and high concentrations of insulin, which promote glucose uptake, appear to counteract any inhibitory action of fatty acids. Therefore, the presence of fatty acids in a normal mixed meal is likely to have little effect on the capacity of the liver to take up, phosphorylate, and oxidize glucose. (C) 2006 Elsevier Inc. All rights reserved.