Fatty acid transport protein 1 (FATP1), a member of the FATP/Slc27 protein family, enhances the cellular uptake of long-chain fatty acids (LCFAs) and is expressed in several insulin-sensitive tissues. In adipocytes and skeletal muscle, FATP1 translocates from an intracellular compartment to the plasma membrane in response to insulin. Here we show that insulin-stimulated fatty acid uptake is completely abolished in FATP1-null adipocytes and greatly reduced in skeletal muscle of FATP1-knockout animals while basal LCFA uptake by both tissues was unaffected. Moreover, loss of FATP1 function altered regulation of postprandial serum LCFA, causing a redistribution of lipids from adipocyte tissue and muscle to the liver, and led to a complete protection from diet-induced obesity and insulin desensitization. This is the first in vivo evidence that insulin can regulate the uptake of LCFA by tissues via FATP1 activation and that FATPs determine the tissue distribution of dietary lipids. The strong protection against diet-induced obesity and insulin desensitization observed in FATP1-null animals suggests FATP1 as a novel antidiabetic target.

Evidence is growing that protease-activated receptor-2 (PAR-2) plays a key role in epithelial inflammation. We hypothesized here that PAR-2 plays a central role in epidermal permeability barrier homeostasis by mediating signaling from serine proteases (SP) in the stratum corneum (SC). Since the SC contains tryptic- and chymotryptic-like activity, we assessed the influence of SP activation/inhibition on barrier function. Acute barrier disruption increases SP activity and blockade by topical SP inhibitors (SPI) accelerates barrier recovery after acute abrogation. This improvement in barrier function is due to accelerated lamellar body (LB) secretion. Since tryptic SP signal certain downstream responses through PAR-2, we assessed its potential role in mediating the negative effects of SP on permeability barrier. Firstly, PAR-2 is expressed in the outer nucleated layers of the epidermis and most specifically under basal condition to the lipid raft (LR) domains. Secondly, tape stripping-induced barrier abrogation provokes PAR-2 activation, as shown by receptor internalization (i.e. receptor movement from LR to cytolpasmic domains). Thirdly, topical applications of PAR-2 agonist peptide, SLIGRL, delay permeability barrier recovery and inhibit LB secretion, while, conversely, PAR-2 knockout mice display accelerated barrier recovery kinetics and enhanced LB secretion, paralleled by increased LR formation and caveolin-1 expression. These results demonstrate first, the importance of SP/SPI balance for normal permeability barrier homeostasis, and second, they identify PAR-2 as a novel signaling mechanism of permeability barrier, that is, of response linked to LB secretion.

In this study we investigated whether hyaluronan (HA)-CD44 interaction influences epidermal structure and function. Our data show that CD44 deficiency is accompanied by reduction in HA staining in CD44 knockout (k/o) mouse skin leading to a marked thinning of epidermis versus wild-type mouse skin. A significant delay in the early barrier recovery (following acute barrier disruption) occurs in CD44 k/o versus wild-type mouse skin. To assess the basis for these alterations in CD44 k/o mouse epidermis, we determined that differentiation markers are greatly reduced in the epidermis of CD44 k/o versus wild-type mice, while conversely HA binding to CD44 triggers differentiation in cultured human keratinocytes. CD44 downregulation (using CD44 small interfering RNAs) also inhibits HA-mediated keratinocyte differentiation. Slower barrier recovery in CD44 k/o mice could be further attributed to reduced lamellar body formation, loss of apical polarization of LB secretion, and downregulation of cholesterol synthesis. Accordingly, HA-CD44 binding stimulates both LB formation and secretion. Together, these observations demonstrate new roles for HA-CD44 interaction in regulating both epidermal differentiation and lipid synthesis/secretion, which in turn influence permeability barrier homeostasis. HA-CD44 signaling could comprise a novel approach to treat skin disorders characterized by abnormalities in differentiation, lipid synthesis, and/or barrier function.

Permeability barrier function is measured with instruments that assess transepidermal water loss (TEWL), either with closed- or open-loop systems. Yet, the validity of TEWL as a measure of barrier status has been questioned recently. Hence, we tested the validity of this measure by comparing TEWL across a wide range of perturbations, with a variety of methods, and in a variety of models. TEWL rates with two closed-chamber systems (VapoMeter and H4300) and one closed-loop system (MEECO) under different experimental in vivo conditions were compared with data from four open-loop instruments, i.e. TM 210, TM 300, DermaLab and EP 1. The instruments were compared in vivo both in humans and hairless mice skin subjected to different degrees of acute barrier disruption. The values obtained with bioengineering systems were correlated with absolute water loss rates, determined gravimetrically. Measurements with both closed and open systems correlated not only with each other, but each method detected different degrees of barrier dysfunction. Although all instruments differentiated among gradations in TEWL in the mid-range of barrier disruption in vivo, differences in very low and very high levels of disruption were less accurately measured with the H4300 and DermaLab systems. Nevertheless, a high Pearson correlation coefficient (r) was calculated for data from all instruments vs. gravimetrically assessed TEWL. Together, these results verify the utility of TEWL as a measure of permeability barrier status. Moreover, all tested instruments are reliable tools for the assessment of variations in permeability barrier function.

Keratinocytes require abundant cholesterol for cutaneous permeability barrier function; hence, the regulation of cholesterol homeostasis is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a pivotal role in regulating cellular cholesterol levels. We demonstrate that ABCA1 is expressed in cultured human keratinocytes (CHKs) and murine epidermis. Liver X receptor (LXR) activation markedly stimulates ABCA1 mRNA and protein levels in CHKs and mouse epidermis. In addition to LXR, activators of peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and retinoid X receptor (RXR), but neither PPARgamma nor retinoic acid receptor, also increase ABCA1 expression in CHKs. Increases in cholesterol supply induced by LDL or mevalonate stimulate ABCA1 expression, whereas inhibiting cholesterol synthesis with statins or cholesterol sulfate decreases ABCA1 expression in CHKs. After acute permeability barrier disruption by either tape-stripping or acetone treatment, ABCA1 expression declines, and this attenuates cellular cholesterol efflux, making more cholesterol available for regeneration of the barrier. In addition, during fetal epidermal development, ABCA1 expression decreases at days 18-22 of gestation (term = 22 days), leaving more cholesterol available during the critical period of barrier formation. Together, our results show that ABCA1 is expressed in keratinocytes, where it is negatively regulated by a decrease in cellular cholesterol levels or altered permeability barrier requirements and positively regulated by activators of LXR, PPARs, and RXR or increases in cellular cholesterol levels.

Many cutaneous disorders are adversely affected by psychological stress (PS), but the responsible mechanisms are poorly understood. Recent studies have demonstrated that PS decreases epidermal proliferation and differentiation, impairs permeability barrier homeostasis, and decreases stratum corneum integrity. PS also increases the production of endogenous glucocorticoids (GC), and both systemic and topical GC cause adverse effects on epidermal structure and function similar to those observed with PS. We therefore hypothesized that increased endogenous GC in PS mediates its adverse cutaneous effects. To test this hypothesis, we used two independent approaches, administering either RU-486, a GC receptor antagonist that inhibits GC action, or antalarmin, a corticotropin-releasing hormone (CRH) receptor antagonist that prevents increased GC production in the face of PS. Inhibition of either GC action or production prevents the PS-induced decline in epidermal cell proliferation and differentiation, impairment in permeability barrier homeostasis, and decrease in stratum corneum (SC) integrity. Moreover, the pathophysiological basis for the abnormality in permeability barrier homeostasis; i.e., decreased lamellar body production and secretion, is restored toward normal by inhibition of GC action. Similarly, the mechanistic basis for the decrease in SC integrity, i.e., a reduction in corneodesmosomes, is also normalized by inhibition of GC action. Thus many of the adverse effects of PS on epidermal structure and function can be attributed to increased endogenous GC and conversely, approaches that either reduce GC production or action might benefit cutaneous disorders that are provoked or exacerbated by PS.

Oxidized cholesterol is present in significant quantities in the typical Western diet. When ingested, oxidized cholesterol is absorbed by the small intestine and incorporated into both chylomicrons and LDL, resulting in LDL that is more susceptible to further oxidation. Feeding studies in animal models and epidemiological studies in humans have suggested that oxidized cholesterol in the diet increases the development of atherosclerosis. In this study, we determined the effect of ezetimibe, a drug that inhibits small intestinal absorption of cholesterol, on the levels of oxidized cholesterol in the serum after a test meal containing oxidized cholesterol. We demonstrate that ezetimibe, 10 mg per day for 1 month, markedly reduced the levels (50% decrease) of oxidized cholesterol in the serum after feeding a test meal containing either alpha-epoxy cholesterol or 7-keto cholesterol, two of the predominant oxidized cholesterols found in the diet. Moreover, the decrease in oxidized cholesterol in the serum was attributable to a decrease in the incorporation of dietary oxidized cholesterol into both chylomicrons and LDL. Because there was no decrease in postprandial triglyceride levels, we conclude that this decrease in oxidized cholesterol levels in the serum is attributable to decreased absorption and not to enhanced clearance. Whether this decrease in oxidized cholesterol absorption prevents or delays the development of atherosclerosis remains to be determined.

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAK(K5 KO)), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAK(K5 KO) mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAK(K5 KO) mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAK(K5 KO) mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK(K5 KO) and control littermates. We found that the critical SC acidification regulator Na(+)/H(+) exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na(+)/H(+) exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells.

Aged skin commonly is afflicted by inflammatory skin diseases or xerosis/eczema that could be triggered or exacerbated by impaired epidermal permeability barrier homeostasis. This defect is linked to reduced epidermal lipid synthesis in humans and in mice of advanced age (i.e., >75 years in human or >18-24 months in mice). We now report that barrier defects in moderately aged humans (50-80 years) or analogously aged mice (12-15 months) are linked instead to defective stratum corneum (SC) acidity. In moderately aged mouse epidermis, we find that abnormal acidification, in turn, is linked to decreased Na+/H+ antiporter (NHE1) expression. Decreased NHE1 levels lead to increased SC pH, which results in defective lipid processing and delayed maturation of lamellar membranes, due to suboptimal activation of the pH-sensitive essential, lipid-processing enzyme, beta-glucocerebrosidase. Conversely, impaired SC integrity in moderately aged mice is due to increased pH-dependent activation of serine proteases, leading to premature degradation of corneodesmosomes. These abnormalities were normalized by exogenously acidifying the SC, suggesting a basis for the well-known acidification therapies that are widely used to treat the pathologic xerosis/eczema seen in moderately aged humans.