39 research outputs found
Role of fatty acid transporters in epidermis: Implications for health and disease
Skin epidermis is an active site of lipid synthesis. The intercellular lipids of human stratum corneum (SC) are unique in composition and quite different from the lipids found in most biological membranes. The three major lipids in the SC are free fatty acids, cholesterol and ceramides. Fatty acids can be synthesized by keratinocytes de novo and, in addition, need to be taken up from the circulation. The latter process has been shown to be protein mediated, and several fatty acid transporters are expressed in skin. Recent studies of transgenic and knockout animal models for fatty acid transporters and the identification of fatty acid transport protein 4 (FATP4 or SLC27A4) mutations as causative for Ichthyosis Prematurity Syndrome highlight the vital roles of fatty acid transport and metabolism in skin homeostasis. This review provides an overview of our current understanding of the role of fatty acids and their transporters in cutaneous biology, including their involvement in epidermal barrier generation and skin inflammation
A Novel Porcine In Vitro Model of the Blood-Cerebrospinal Fluid Barrier with Strong Barrier Function
Epithelial cells of the plexus choroideus form the structural basis of the blood-cerebrospinal fluid barrier (BCSFB). In vitro models of the BCSFB presenting characteristics of a functional barrier are of significant scientific interest as tools for examination of BCSFB function. Due to a lack of suitable cell lines as in vitro models, primary porcine plexus epithelial cells were subjected to a series of selective cultivation steps until a stable continuous subcultivatable epithelial cell line (PCP-R) was established. PCP-R cells grow in a regular polygonal pattern with a doubling time of 28–36 h. At a cell number of 1.5×105 in a 24-well plate confluence is reached in 56–72 h. Cells are cytokeratin positive and chromosomal analysis revealed 56 chromosomes at peak (84th subculture). Employing reverse transcription PCR mRNA expression of several transporters and components of cell junctions could be detected. The latter includes tight junction components like Claudin-1 and -3, ZO-1, and Occludin, and the adherens junction protein E-cadherin. Cellular localization studies of ZO-1, Occludin and Claudin-1 by immunofluorescence and morphological analysis by electron microscopy demonstrated formation of a dense tight junction structure. Importantly, when grown on cell culture inserts PCP-R developed typical characteristics of a functional BCSFB including high transepithelial electrical resistance above 600 Ω×cm2 as well as low permeability for macromolecules. In summary, our data suggest the PCP-R cell line as a suitable in vitro model of the porcine BCSFB
Characteristics of Oleate Binding to Liver Plasma-Membranes and its Uptake by Isolated Hepatocytes
To clarify mechanisms of hepatic free fatty acid uptake, [3H]oleate uptake by isolated rat hepatocytes was studied,
using solutions of 150 laM bovine serum albumin at oleate:albumin molar ratios of 0.033-6.7:1. Oleate partitioning
between liver plasma membranes and albumin was also studied, and used to ascertain the membrane binding function
for oleate. The experimental uptake curve was complex, but could be resolved by computer fitting into a sum of two
components, one a saturable and the second a linear function of the unbound oleate concentration. The saturable
component comprises > 90% of total oleate uptake when the oleate:albumin molar ratio is < 2.5, but < 50% when
this ratio is > 5. Membrane binding also consisted of a sum of a saturable and a linear component. By comparison
of the computer-fitted uptake and binding functions, separate rate constants for the transfer into the cell of the
saturably and non-saturably bound oleate were estimated to be 0.7 s -~ and 0.05 s -~, respectively. The former is
compatible with a specific, protein-mediated process. It is 15-times greater than the corresponding rate constant for
transfer of non-saturably bound oleate into the cell, which in turn is similar to reported rates of non-specific 'flip-flop'
of fatty acids across lipid bilayers. The observed kinetics are not consistent with models in which uptake occurs
principally from the albumin-bound pool of oleate, or solely from the oleate which has partitioned passively into the
lipid bilayer of the plasma membrane
The Hepatocellular Uptake of Free Fatty-Acids Is Selectively Preserved During Starvation
Background/Aims: The liver loses protein during fasting. This study sought to determine if hepatic protein loss during fasting selectively preserves functions important to survival such as uptake of fatty acids, which are major energy substrates in that condition. Methods: initial [H-3]oleate uptake and efflux rates in hepatocytes from starved (for 48 hours) and fed male rats were measured in media containing 250 mu mol/L albumin at oleate/albumin ratios of 0.2:1-2:1. Uptake rates of sulfobromophthalein, taurocholate, and glucose were also determined. Results: Initial oleate uptake rate was saturable with respect to unbound oleate concentration. Maximum initial velocity expressed per cell number did not differ between fasted and fed animals, but measured cell volume and estimated surface area were decreased in starved vs. fed hepatocytes (921 +/- 21 vs. 1623 +/- 58 mu m(2), respectively; P< 0.001). Consequently, when expressed per surface area, maximum initial velocity was greater in starved cells (17 +/- 3 vs. 10 +/- 2 [pmol min(-1) mu m(2)] x 10(-7); P < 0.02). Expressed similarly, oleate efflux was also greater from starved hepatocytes and was inhibited by an antibody to plasma membrane fatty acid binding protein (FABP(pm)). FABP(pm) concentration per unit area of plasma membrane also increased in starved hepatocytes (P < 0.05). By contrast, uptake rates of sulfobromophthalein, taurocholate, and glucose by starved hepatocytes were decreased when expressed per cell number and unchanged per unit area. Conclusions: During fasting, the hepatocellular uptake mechanism for oleate is selectively preserved compared with those for sulfobromophthalein, taurocholate, or glucos
Oleate uptake by isolated hepatocytes and the perfused rat liver is competitively inhibited by palmitate
Competition for uptake between long-chain free fatty acids has been difficult to document, because there has been no algorithm for computing unbound concentrations of two fatty acids simultaneously in solution with albumin. We modified an iterative procedure to permit this computation and studied initial [ 3H]oleate uptake by isolated hepatocytes and steady-state uptake by the single-pass perfused rat liver from 600 \u3bcM bovine serum albumin solutions containing various concentrations of oleate in the presence and absence of palmitate. In both systems, the Michaelis-Menten constant was significantly higher in the presence of palmitate than in its absence, whereas the maximal reaction velocity was unaltered, indicating competitive inhibition. In additional experiments employing the multiple transhepatic indicator-dilation technique, the influx rate constant and permeability- surface area product for oleate influx were significantly reduced by palmitate, confirming that the competition observed in the conventional perfused liver studies was at the influx step. Long-chain fatty acid uptake has now been shown to exhibit all the kinetic properties of facilitated transport and cannot be attributed solely to passive diffusion
Long-chain fatty acid uptake is upregulated in omental adipocytes from patients undergoing bariatric surgery for obesity
Long-chain fatty acid uptake is upregulated in omental adipocytes from patients undergoing bariatric surgery for obesity
OBJECTIVE: To determine the impact of obesity on adipocyte cell size and long-chain fatty acid (LCFA) uptake kinetics in human subjects undergoing laparoscopic abdominal surgery. SUBJECTS: A total of 10 obese patients (BMI 49.8±11.9 (s.d.) kg/m2) undergoing laparoscopic bariatric surgery, and 10 nonobese subjects (BMI 24.2±2.3 kg/m2) undergoing other clinically indicated laparoscopic abdominal surgical procedures. MEASUREMENTS: Cell size distribution and [3H]oleic acid uptake kinetics were studied in adipocytes isolated from omental fat biopsies obtained during surgery. Adipocyte surface area (SA) was calculated from the measured cell diameters. Plasma leptin and insulin concentrations were measured by RIA in fasting blood samples obtained on the morning of surgery. RESULTS: The mean SA of obese adipocytes (41 508±5381 μ2/cell) was increased 2.4-fold compared to that of nonobese adipocytes (16928±6529 μ2/cell; P < 0.01). LCFA uptake in each group was the sum of saturable and nonsaturable components. Both the Vmax of the saturable component (21.3±6.3 vs 5.1±1.9 pmol/s/50000 cells) and the rate constant k of the nonsaturable component (0.015±0.002 vs 0.0066±0.0023 ml/s/50 000 cells) were increased (P < 0.001) in obese adipocytes compared with nonobese controls. When expressed relative to cell size, Vmax/μ2 SA was greater in obese than nonobese adipocytes (P < 0.05), whereas k/μ2 SA did not differ between the groups. CONCLUSION: The data support the concepts that (1) adipocyte LCFA uptake consists of distinct facilitated (saturable) and diffusive processes; (2) increased saturable LCFA uptake in obese adipocytes is not simply a consequence of increased cell size, but rather reflects upregulation of a facilitated transport process; and (3) the permeability of adipocyte plasma membranes to LCFA is not appreciably altered by obesity, and increased nonsaturable uptake in obese adipocytes principally reflects an increase in cell SA. Regulation of saturable LCFA uptake by adipocytes may be an important control point for body adiposity
Insulin- and leptin-regulated fatty acid uptake plays a key causal role in hepatic steatosis in mice with intact leptin signaling but not in ob/ob or db/db mice
Hepatic steatosis results from several processes. To assess their relative roles, hepatocellular long-chain fatty acid (LCFA) uptake was assayed in hepatocytes from C57BL/6J control mice, mice with steatosis from a high-fat diet (HFD) or 10%, 14%, or 18% ethanol (EtOH) in drinking water [functioning leptin-signaling groups (FLSGs)], and ob/ob and db/db mice. Vmax for uptake was increased vs. controls (P < 0.001) and correlated significantly with liver weight and triglycerides (TGs) in all FLSG mice but was minimally or not increased in ob/ob and db/db mice, in which liver weights and TGs greatly exceeded projections from regressions in FLSG animals. Coefficients of determination (R2) for these FLSG regressions suggest that increased LCFA uptake accounts for ∼80% of the increase in hepatic TGs within these groups, but increased lipogenic gene expression data suggest that enhanced LCFA synthesis is the major contributor in ob/ob and db/db. Got2, Cd36, Slc27a2, and Slc27a5 gene expression ratios were significantly upregulated in the EtOH groups, correlating with sterol regulatory element binding protein 1c (SREBP1c) and Vmax, but only Cd36 expression was increased in HFD, ob/ob, and db/db mice. Comparison of Vmax with serum insulin and leptin suggests that both hormones contribute to upregulation of uptake in the FLSG animals. Thus, increased LCFA uptake, reflecting SREBP1c-mediated upregulation of four distinct transporters, is the dominant cause of steatosis in EtOH-fed mice. In ob/ob and db/db mice, increased LCFA synthesis appears more important. In FLSG animals, insulin upregulates hepatocellular LCFA uptake. Leptin appears to upregulate LCFA uptake or to be essential for full expression of upregulation by insulin