The etiology of atherosclerosis is complex and multifactorial but there is extensive evidence indicating that oxidized lipoproteins may play a key role. At present, the site and mechanism by which lipoproteins are oxidized are not resolved, and it is not clear if oxidized lipoproteins form locally in the artery wall and/or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on demonstrating that such potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in the diet. Thus, the purpose of our work has been to determine in humans whether oxidized dietary oxidized fats such as oxidized fatty acids and oxidized cholesterol are absorbed and contribute to the pool of oxidized lipids in circulating lipoproteins. When a meal containing oxidized linoleic acid was fed to normal subjects, oxidized fatty acids were found only in the postprandial chylomicron/chylomicron remnants (CM/RM) which were cleared from circulation within 8 h. No oxidized fatty acids were detected in low density lipoprotein (LDL) or high density lipoprotein (HDL) fractions at any time. However, when alpha-epoxy cholesterol was fed to human subjects, alpha-epoxy cholesterol in serum was found in CM/RM and also in endogenous very low density lipoprotein, LDL, and HDL and remained in the circulation for 72 h. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. We have suggested that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into CM/RM fraction and then transferred to LDL and HDL contributing to lipoprotein oxidation. We hypothesize that diet-derived oxidized fatty acids in chylomicron remnants and oxidized cholesterol in remnants and LDL accelerate atherosclerosis by increasing oxidized lipid levels in circulating LDL and chylomicron remnants. This hypothesis is supported by our feeding experiments in animals. When rabbits were fed oxidized fatty acids or oxidized cholesterol, the fatty streak lesions in the aorta were increased by 100%. Moreover, dietary oxidized cholesterol significantly increased aortic lesions in apo-E and LDL receptor-deficient mice. A typical Western diet is rich in oxidized fats and therefore could contribute to the increased arterial atherosclerosis in our population.

In neonatal rat stratum corneum (SC), pH declines from pH 6.8 at birth to adult levels (pH 5.0-5.5) over 5-6 d. Liver X receptor (LXR) activators stimulate keratinocyte differentiation, improve permeability barrier homeostasis, and accelerate the in utero development of the SC. In this manuscript we determined the effect of LXR activators on SC acidification in the neonatal period and whether these activators correct the functional abnormalities in permeability barrier homeostasis and SC integrity/cohesion. Formation of the acid SC-buffer system was accelerated by topically applying the LXR activator, 22(R)-hydroxycholesterol, and non-oxysterol activators of LXR, TO-901317, and GW-3965. A sterol which does not activate LXR had no effect. LXR activation increased secretory phospholipase A(2) (sPLA(2)) activity and conversely, inhibition of sPLA(2) activity prevented the LXR induced increase in SC acidification, suggesting that increasing sPLA(2) accounts in part, for the LXR stimulation of acidification. LXR activation resulted in an improvement in permeability barrier homeostasis, associated with an increased maturation of lamellar membranes attributable to an increased beta-glucocerebrosidase activity. SC integrity cohesion also normalized in LXR-activator-treated animals and was associated with an increase in corneodesmosomes and in desmoglein 1 expression. These results demonstrate that LXR activators stimulate the formation of an acidic SC and improve both permeability barrier homeostasis and SC integrity/cohesion.

Epidermis and sebocyte-derived lipids are derived both from de novo synthesis and through uptake of fatty acids from the circulation. Plasma membrane proteins can significantly contribute to the latter process. In particular, fatty acid transport proteins (FATP/solute carrier family 27) are integral transmembrane proteins that enhance the uptake of long-chain fatty acids into cells. Using specific antisera against all six mammalian FATP, we found that both human and mouse skin express FATP1, -3, -4, and -6. In adult skin, FATP1 and -3 are expressed predominantly by keratinocytes, whereas FATP4 is strongly expressed by sebaceous glands and FATP6 by hair follicle epithelia. Sustained barrier disruption leads to increases in FATP1 and -6 levels as well as a robust increase in CD36 protein. Notably, expression of FATP1 by embryonic keratinocytes at day 18.5 was lower, and FATP4 increased in comparison with adult epidermis. Together, these findings indicate that FATP are not only expressed by different cell types within the skin, but also that their localization is dynamically regulated during development.

Previously, we demonstrated that topical applications of peroxisome proliferator-activated receptors (PPARs) and liver X receptor (LXR) activators improve permeability barrier homeostasis. We showed further that stimulation of epidermal differentiation provides one mechanism that could account for such improvement. Here, we studied the effects of these agents on the lipid matrix of the stratum corneum. Hairless mice were treated topically with activators of PPARalpha (WY14643), PPARdelta (GW1514), PPARgamma (ciglitazone), and LXR (22(R)-cholesterol or TO901317) or vehicle twice daily for 3 days. All activators significantly increased epidermal cholesterol, fatty acid, and sphingolipid synthesis, including the production of barrier-specific ceramide species. In addition, lamellar body (LB) formation, secretion, and post-secretory processing accelerated significantly following acute barrier disruption in PPAR/LXR-activator-treated animals. Finally, the activity of epidermal beta-glucocerebrosidase, a key lipid-processing enzyme, increased in PPAR/LXR-activator-treated animals. Thus, topical PPAR and LXR activators stimulate epidermal lipid synthesis, increase LB secretion, and accelerate extracellular lipid processing, providing additional mechanisms that further account for their ability to improve epidermal permeability barrier homeostasis. Since the liposensors are activated by endogenous lipid metabolites, they may serve as unique regulators of barrier homeostasis.

In a murine model of epidermal hyperplasia reproducing some of the abnormalities of several common skin disorders, we previously demonstrated the antiproliferative and pro-differentiating effects of peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and liver X receptor activators. Unlike other subgroups of PPAR activators, thiazolidinediones (TZDs), a family of PPARgamma ligands, did not inhibit keratinocyte proliferation in normal murine skin. Here, we studied the effects of two TZDs, namely ciglitazone (10 mM) and troglitazone (1 mM), in the same murine model where epidermal hyperproliferation was reproduced by repeated barrier abrogation with tape stripping. Topical treatment with ciglitazone and troglitazone resulted in a marked and significant decrease in epidermal thickness. Furthermore, in all TZD-treated groups, we observed a significant decrease in keratinocyte proliferation using proliferating cell nuclear antigen, 5-bromo-2'-deoxyuridine, and tritiated thymidine incorporation. However, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, we found no difference in apoptosis between different treatments, emphasizing that it is the antiproliferative role of these activators that accounts for the decrease of epidermal thickness. Finally, using immunohistochemical methods, we determined the effects of ciglitazone on keratinocyte differentiation in this hyperproliferative model. We observed an increased expression of involucrin and filaggrin following ciglitazone treatment, suggesting a pro-differentiating action of TZDs in this model. In summary, topical TZDs significantly reduce epidermal keratinocyte proliferation while promoting differentiation in a murine model of hyperproliferative epidermis. Together, these results suggest that in addition to their metabolic effects currently in use in the treatment of type 2 diabetes, topical TZDs could be considered as potential alternative therapeutic agents in hyperproliferative skin diseases such as psoriasis.

Mutations in the SPINK5 gene encoding the serine protease (SP) inhibitor, lymphoepithelial-Kazal-type 5 inhibitor (LEKTI), cause Netherton syndrome (NS), a life-threatening disease, owing to proteolysis of the stratum corneum (SC). We assessed here the basis for phenotypic variations in nine patients with "mild", "moderate", and "severe" NS. The magnitude of SP activation correlated with both the barrier defect and clinical severity, and inversely with residual LEKTI expression. LEKTI co-localizes within the SC with kallikreins 5 and 7 and inhibits both SP. The permeability barrier abnormality in NS was further linked to SC thinning and proteolysis of two lipid hydrolases (beta-glucocerebrosidase and acidic sphingomyelinase), with resultant disorganization of extracellular lamellar membranes. SC attenuation correlated with phenotype-dependent, SP activation, and loss of corneodesmosomes, owing to desmoglein (DSG)1 and desmocollin (DSC)1 degradation. Although excess SP activity extended into the nucleated layers in NS, degrading desmosomal mid-line structures with loss of DSG1/DSC1, the integrity of the nucleated epidermis appears to be maintained by compensatory upregulation of DSG3/DSC3. Maintenance of sufficient permeability barrier function for survival correlated with a compensatory acceleration of lamellar body secretion, providing a partial permeability barrier in NS. These studies provide a mechanistic basis for phenotypic variations in NS, and describe compensatory mechanisms that permit survival of NS patients in the face of unrelenting SP attack.

CONTEXT

Activation of peroxisome proliferator-activated receptor-gamma by thiazolidinediones (TZDs) results in lower bone mass in mice.

OBJECTIVE

The objective of the study was to determine whether TZD use is associated with changes in bone mineral density (BMD) in older adults with type 2 diabetes.

DESIGN

We analyzed 4-yr follow-up data from the Health, Aging, and Body Composition observational study.

SETTING

The study was conducted in a general community.

PATIENTS

White and black, physically able men and women, aged 70-79 yr at baseline with diabetes defined by self-report, use of hypoglycemic medication, elevated fasting glucose (>/=126 mg/dl), or elevated 2-h glucose tolerance test (>/=200 mg/dl) participated in the study.

MAIN OUTCOME MEASURES

Whole-body, lumbar spine (derived from whole body), and hip BMD were measured by dual-energy x-ray absorptiometry at 2-yr intervals.

RESULTS

Of 666 diabetic participants, 69 reported TZD use at an annual visit, including troglitazone (n = 22), pioglitazone (n = 30), and/or rosiglitazone (n = 31). Those with TZD use had higher baseline hemoglobin A(1c) and less weight loss over 4 yr but similar baseline BMD and weight than others with diabetes. In repeated-measures models adjusted for potential confounders associated with TZD use and BMD, each year of TZD use was associated with greater bone loss at the whole body [additional loss of -0.61% per year; 95% confidence interval (CI) -1.02, -0.21% per year], lumbar spine (-1.23% per year; 95% CI -2.06, -0.40% per year), and trochanter (-0.65% per year; 95% CI -1.18, -0.12% per year) in women, but not men, with diabetes.

CONCLUSION

These observational results suggest that TZDs may cause bone loss in older women. These results need to be tested in a randomized trial.

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.