Optimal nutrition and the ever-changing dietary landscape: a conference report.

The field of nutrition has evolved rapidly over the past century. Nutrition scientists and policy makers in the developed world have shifted the focus of their efforts from dealing with diseases of overt nutrient deficiency to a new paradigm aimed at coping with conditions of excess-calories, sedentary lifestyles and stress. Advances in nutrition science, technology and manufacturing have largely eradicated nutrient deficiency diseases, while simultaneously facing the growing challenges of obesity, non-communicable diseases and aging. Nutrition research has gone through a necessary evolution, starting with a reductionist approach, driven by an ambition to understand the mechanisms responsible for the effects of individual nutrients at the cellular and molecular levels. This approach has appropriately expanded in recent years to become more holistic with the aim of understanding the role of nutrition in the broader context of dietary patterns. Ultimately, this approach will culminate in a full understanding of the dietary landscape-a web of interactions between nutritional, dietary, social, behavioral and environmental factors-and how it impacts health maintenance and promotion.

Regulation of human immune gene expression as influenced by a commercial blended Echinacea product: preliminary studies.

Consumption of Echinacea at the first sign of symptoms has been clinically shown to reduce both the severity and duration of cold and flu. Quantitative polymerase chain reaction optimized for precision and reproducibility was utilized to explore in vitro and in vivo changes in the expression of immunomodulatory genes in response to Echinacea. In vitro exposure of THP-1 cells to 250 microg/ml of Echinacea species extracts induced expression (up to 10-fold) of the interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, interleukin-8, and interleukin-10 genes. This preliminary result is consistent with a general immune response and activation of the nonspecific immune response cytokines. In vivo gene expression within peripheral leukocytes was evaluated in six healthy nonsmoking subjects (18-65 years of age). Blood samples were obtained at baseline and on Days 2, 3, 5, and 12 after consuming a commercial blended Echinacea product, three tablets three times daily (1518 mg/day) for two days plus one additional dose (506 mg) on day three. Serum chemistry and hematological values were not different from baseline, suggesting that liver or bone marrow responses were not involved in acute responses to Echinacea. The overall gene expression pattern at 48 hr to 12 days after taking Echinacea was consistent with an antiinflammatory response. The expression of interleukin-1beta, tumor necrosis factor-alpha, intracellular adhesion molecule, and interleukin-8 was modestly decreased up through Day 5, returning to baseline by day 12. The expression of interferon-alpha steadily rose through Day 12, consistent with an antiviral response. These preliminary data present a gene expression response pattern that is consistent with Echinacea's reported ability to reduce both the duration and intensity of cold and flu symptoms.

Cloning and characterization of retinol dehydrogenase transcripts expressed in human epidermal keratinocytes.

The normal growth and differentiation of the epidermis require an adequate supply of vitamin A. The active form of vitamin A for normal epidermal homeostasis is retinoic acid (RA). Retinoic acid controls the expression of retinoid-responsive genes via interactions of the retinoic acid/nuclear receptor complexes at specific DNA sequences in their control regions. The message conveyed by RA is likely modulated by the concentration of the ligand available for binding to the receptors. Following the uptake of plasma retinol, epidermal keratinocytes synthesize retinoic acid via two sequential reactions with retinaldehyde as an intermediate. Several retinol dehydrogenase (RDH) enzymes, members of the short-chain dehydrogenase/reductase (SDR) gene superfamily, catalyze the first and rate-limiting step that generates retinaldehyde from retinol bound to cellular retinol-binding protein (holo-CRBP). However, little is known about these enzymes and their genes in the epidermal cells. Our work describes the first member of the RDH family found in epidermis. We show that this gene is expressed predominantly in the differentiating spinous layers and that it is under positive, feed-forward regulation by retinoic acid. It encodes a protein that, using NAD+ as a preferred cofactor, utilizes free and CRBP-bound all-trans-retinol and steroids as substrates.

Dermal fibroblasts actively metabolize retinoic acid but not retinol.

The accessibility of plasma retinol and retinoic acid to the epidermis may be influenced by the number and metabolic capacity of fibroblasts in papillary dermis. The metabolism of retinol-binding protein-bound all-trans-retinol, and albumin-bound all-trans-retinoic acid, by fibroblasts cultured on plastic dishes or in type I collagen gels, was examined. There were no significant differences in the metabolism of either retinoid by fibroblasts as a function of culture condition. There were large differences between retinoids, however. Retinoic acid was rapidly taken up and metabolized to unidentified polar metabolites that were released to the medium. Metabolic capacity was not saturated up to a medium retinoic acid concentration of 1 microM, and was induced further by prior exposure to retinoic acid. In contrast, retinol, although readily taken up, was not metabolized, i.e., neither retinoic acid nor retinyl ester was formed. By immunohistochemistry, the average number of fibroblasts in a 100 microm thickness of papillary dermis was estimated to be 1 x 10(6) cells per cm2. Utilizing this value, the capacity of dermal fibroblasts to metabolize retinoic acid based on fibroblast abundance in the dermis was calculated. The results suggest that fibroblasts could limit delivery of plasma retinoic acid but not retinol to the epidermis on the basis of their metabolic capacity and abundance in the dermis.

Differentiation of human epidermal keratinocytes is accompanied by increased expression of CRABP-II and increased cellular concentration of retinoic acids: retention of newly synthesized retinoic acids by CRABP-II.

Keratinocytes differentiating in vitro exhibit greater cytosolic capacity for retinoic acid synthesis from retinol or retinaldehyde as compared to nondifferentiated cells (Siegenthaler et al. 1990. Biochem. J. 268: 371-378), and increased expression of CRABP-II (Siegenthaler et al. 1988. Exp. Cell Res. 178: 114-126). Based on these observations, the content and disposition of [3H]retinoic acids were determined in intact, nondifferentiated and differentiating keratinocytes incubated with [3H]retinaldehyde or [3H]retinol. Differentiating keratinocytes contained higher levels of [3H] retinoic acids compared to undifferentiated cells when either [3H]retinaldehyde or [3H]retinol was the substrate. The largest increases in [3H]retinoic acids were achieved with [3H]retinaldehyde. Differentiation-associated increases in [3H]retinoic acids correlated with cellular content of retinoid alcohol substrates in incubations with retinaldehyde but not in incubations with retinol. Consistent with previous observations, CRABP-II was significantly increased in differentiating cells. Moreover, newly synthesized [3H]retinoic acids were retained within cells bound to CRABP-II. The results suggest that increasing cellular concentration of retinoic acids in in vitro differentiating keratinocytes is achieved by a combination of increased activity of the retinoic acid synthesis pathway and increased cellular content of CRABP-II.

Metabolism of all-trans-retinoic acid by cultured human epidermal keratinocytes.

The uptake and metabolism of exogenous all-trans-retinoic acid by human epidermal keratinocytes in submerged culture was examined. Retinoic acid presented to keratinocytes in physiological form bound to albumin was rapidly taken up. Peak cellular concentrations were achieved within 1 h and were 50- to 100-fold higher than that in medium. Retinoic acid metabolism was less rapid but was vigorous, exhibiting a half-life of 6 h. Neither uptake nor metabolism was saturable at medium retinoic acid concentrations up to 1 microM. Eighty-five percent of retinoic acid was metabolized to unidentified polar compounds which were excreted from cells to the medium. The production and clearance of the polar metabolites was inhibited 60% by 10 microM ketoconazole. Fifteen percent of intracellular retinoic acid was converted to 3,4-didehydroretinoic acid, was proportional to cellular retinoic acid concentration, and was not affected by ketoconazole. Cellular retinyl ester content, which is sensitive to exogenous retinoic acid, was correlated with both the concentration and the quantity, of retinoic acid in the culture system. These results suggest that the metabolism of retinoic acid in keratinocytes is substrate limited and has potential to limit the availability of exogenous retinoic acid for retinoid signaling.

all-trans-retinoic acid regulates retinol and 3,4-didehydroretinol metabolism in cultured human epidermal keratinocytes.

The potential for all-trans-retinoic acid to regulate the metabolism of 3H-retinol and 3H-3,4-didehydroretinol was examined in cultured human epidermal keratinocytes. Confluent cultures were treated daily with medium containing 5% fetal bovine serum or the same medium supplemented with nanomolar concentrations of all-trans-retinoic acid for up to 3 d. During the last 24 of treatment, cells were incubated with 3H-retinol or 3H-3,4-didehydroretinol for 24 h (isotopic steady state) to label the endogenous retinoids. After the labeling period, one group of cells was harvested and another group was allowed to incubate for an additional 24 h in the absence of medium retinol for the determination of endogenous 3H-retinoid utilization. The 3H-retinoids present in cells were extracted and quantitated by reverse-phased high-pressure liquid chromatography. Keratinocytes treated with retinoic acid and labeled with 3H-retinol exhibited time- and concentration-dependent (i) increases in retinyl ester mass, (ii) increases in the rate of retinyl ester synthesis, (iii) decreases in retinyl ester utilization, and (iv) decreases in the cellular concentrations of retinoic and 3,4-didehydroretinoic acids. There was no effect of exogenous retinoic acid on its own metabolism. Cells labeled with 3H-3,4-didehydroretinol exhibited exclusive labeling of vitamin A2-related retinoids suggesting that the A1 to A2 conversion is not reversible. Treatment of cells with low nanomolar concentrations of retinoic acid decreased the utilization of 3,4-didehydroretinyl esters, decreased the production of 3,4-didehydroretinoic acid but had no effect on the synthesis of 3,4-didehydroretinol or its esters. The results demonstrate that keratinocytes respond to extracellular retinoic acid by decreasing endogenous production of active retinoids, sequestering extracellular substrate retinol as retinyl ester, and decreasing ester utilization.

Metabolic regulation of active retinoid concentrations in cultured human epidermal keratinocytes by exogenous fatty acids.

Retinol metabolism was examined in cultured human epidermal keratinocytes incubated with medium containing physiological concentrations of bovine serum albumin-free fatty acid complexes and [3H]retinol. Unsaturated 16- and 18-carbon fatty acids: (1) promoted up to a 50% increase in the steady state total cell retinoid mass which was accounted for as retinyl ester corresponding to the added fatty acid, (2) decreased the rate of endogenous retinyl ester utilization up to 80%, (3) decreased the steady state cellular concentrations of retinol, 3,4-didehydroretinol and their respective carboxylic acids up to 80%, and (4) did not alter the rate of retinoic acid metabolism. Increasing the medium retinol concentration augmented the fatty acid-stimulated increase in esters and blunted the extent of decrease in alcohols and acids. Saturated fatty acids with 14, 16, 17, and 18 carbons also reduced the rate of retinyl ester utilization and the cellular concentrations of the retinoid alcohols and acids but had little to no effect on retinyl ester synthesis. The data demonstrate that exogenous fatty acids possess the capacity to regulate the cellular concentrations of substrate retinoid alcohols, active retinoid acids, and the overall rate of retinol metabolism via substrate-mediated stimulation of retinol esterification and decreasing retinyl ester utilization. Fatty acids thus possess the potential to play a physiological role in modulating retinoid signaling in keratinocytes by regulating cellular concentrations of active retinoids.

Characterization of retinol metabolism in cultured human epidermal keratinocytes.

Retinoids present in cultured human epidermal keratinocytes maintained in medium containing 25 nM retinol were identified and quantitated utilizing reverse-phase gradient high performance liquid chromatography. Total cell retinoid mass and composition averaged 12.59 pmol/mg cell protein +/- 1.86, (mean +/- S.D., n = 5) between strains and was constant with culture age. Long chain fatty acid esters of retinol and 3,4-didehydroretinol comprised greater than 95% of total retinoid, each contributing 77 and 23% of total esters, respectively. In mature stratified cultures, less abundant retinoids included unesterified retinol, 1%, and 3,4-didehydroretinol, 0.5%, retinoic acid, 0.5%, and a retinoid not previously reported in mammalian cells, 3,4-didehydroretinoic acid, 0.3%. This absolute and relative retinoid composition was essentially constant with culture age except that 3,4-didehydroretinoic acid was not detected in 3-4-day-old cultures (16-32 cell colonies), suggesting that keratinocyte differentiation may be related to endogenous changes in production of "active" retinoids. Separate pools of retinyl and 3,4-didehydroretinyl esters served as predominant sources of substrate for production of retinoic and 3,4-didehydroretinoic acids, respectively, suggesting that their production may be regulated independently.

Vitamin A status regulates hepatic lecithin: retinol acyltransferase activity in rats.

The activity of lecithin:retinol acyltransferase (LRAT) was determined in microsomes from the liver and small intestine of rats with differing vitamin A status. In animals depleted of retinol, as judged by undetectable liver vitamin A stores and low plasma retinol concentrations, hepatic LRAT activity was almost undetectable, whether assayed with retinol bound to cellular retinol-binding protein or solvent-dispersed retinol. In contrast, neither the activity of intestinal LRAT nor that of acyl-CoA:retinol acyltransferase in either liver or intestine differed from that of vitamin A-adequate rats. During the course of vitamin A depletion, liver LRAT activity fell progressively, nearly in parallel to the decrease in plasma retinol concentration. Oral repletion of vitamin A-depleted rats with 0.8 mg of retinol resulted in a very rapid restoration of plasma retinol concentration and full recovery of hepatic LRAT activity within 24 h, together with deposition of retinyl ester in the liver. These data strongly implicate LRAT activity in liver as responsible for the storage of hepatic retinyl esters. Retention of the intestine's capacity to esterify retinol during vitamin A deficiency provides a mechanism for capture of dietary vitamin A, while reduced hepatic LRAT activity may function to redirect retinol in liver from storage to other metabolic pathways.

Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes.

Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consistent with lecithin:retinol acyltransferase (LRAT) activity. LRAT activity exhibited the same Km (2 microM retinol) between tissues but a higher Vmax in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg microsome protein, respectively). Solvent-dispersed retinol was also esterified in a fatty acyl CoA-dependent, PMSF-resistant reaction, consistent with acyl CoA:retinol acyltransferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity displayed a similar Vmax (300 pmol/min/mg microsome protein) between tissues but Km values of 15 and 5 microM for retinol and fatty acyl CoA in mammary gland as compared to 30 and 25 microM, respectively, in the liver. Thus, when substrate was near or below Km, retinol esterification occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concentration of CRBP in the cytosol, determined by Western blotting, was approximately 2 microM in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esterification is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver.

Regulation of retinol uptake and esterification in MCF-7 and HepG2 cells by exogenous fatty acids.

The influence of extracellular fatty acids on the uptake and esterification of [3H]retinol bound to human retinol-binding protein (RBP), to RBP-transthyretin (TTR), or in dispersed form by the human hepatoma, HepG2, and human mammary epithelial carcinoma, MCF-7, cell lines was studied. The esterification of [3H]retinol was significantly increased in cells incubated with myristic, palmitic, stearic oleic, or linoleic acid-albumin complexes and was observed for all forms of [3H]retinol. Enhancement of [3H]retinol uptake was also observed in cells incubated with these fatty acids, but this increase was relatively small for the dispersed form as compared to that observed for [3H]retinol bound to RBP or RBP-TTR. Comparing equal concentrations of the [3H]retinol donors, cell uptake and esterification was greatest from the dispersed form and least from that bound to RBP-TTR. After preincubation of cells with oleate, uptake and esterification of [3H]retinol was increased but not to the extent observed when oleate and [3H]retinol donor were co-incubated. Incubation of cells with oleate resulted in rapid and correlated increases in the rates of [3H]retinol uptake and esterification which persisted until the steady state for [3H]retinol uptake was achieved. Beyond this time, net esterification of [3H]retinol continued in the presence of oleate. This kinetic pattern was observed for all [3H]retinol donors. These effects on [3H]retinol uptake and esterification were dose-dependent as the oleate to albumin ratio was varied from 0.5 to 3.0 and were observed across a physiological concentration range of RBP-3H-retinol. The data indicate that: 1) the fatty acid status of cells is a determinant of retinol uptake and esterification; and 2) the form of retinol presentation to cells is not qualitatively important for these processes.

Relationship of plasma lipoproteins and the monocyte-macrophage system to atherosclerosis severity in cholesterol-fed pigeons.

The purpose of this study was to determine if there was a relationship between plasma beta-VLDL concentrations and atherosclerosis severity in cholesterol-fed pigeons, and if so, whether this correlated with cholesterol accumulation in tissues rich in cells of the monocyte-macrophage system (liver, spleen, peritoneal macrophages). Among individual birds consuming a cholesterol-containing diet, plasma beta-VLDL concentrations ranged from 14 to 1979 mg/dl. In these animals the cholesterol content of peritoneal macrophages, liver, and spleen was positively correlated with plasma concentrations of beta-VLDL and LDL, but not with HDL. When added in vitro to peritoneal macrophages from control pigeons, beta-VLDL stimulated a nearly 200-fold increase in cholesterol accumulation, but there was no relationship between the ability of beta-VLDL to stimulate macrophage cholesterol accumulation and the extent of atherosclerosis in the animal from which the beta-VLDL was obtained. Cholesterol feeding for up to 6 months increased the severity of atherosclerosis in both atherosclerosis-susceptible White Carneau and resistant Show Racer pigeons, but there was no correlation of atherosclerosis severity with beta-VLDL or HDL concentrations, and only a weak relationship with LDL concentrations. The results are consistent with the conclusion that atherosclerosis susceptibility in pigeons cannot be explained by quantitative or qualitative differences in plasma beta-VLDL. Instead, differences in susceptibility are probably mediated at the level of the arterial wall, perhaps by genetic differences that influence the way an individual animals' arterial cells (endothelial, smooth muscle, macrophages) interact with specific plasma lipoproteins.

Exchange and mass efflux of cholesterol in macrophages. Evidence for a common mechanism and a role for plasma membrane proteins.

Exchange and net mass efflux of cholesterol were investigated in [3H]cholesterol-labeled or cholesteryl ester-loaded murine peritoneal macrophages, respectively. Macrophages were subjected to mild proteolysis prior to measurements of mass efflux or exchange to assess whether plasma membrane proteins participated in either process. Cholesterol exchange and mass efflux were inhibited up to 70% following trypsinization. The inhibitory effect was reversible as cells regained normal efflux and exchange 6-8 hr following treatment. Incubation of trypsinized cells with cycloheximide prevented recovery, indicating that protein synthesis was necessary for restoration of normal cholesterol efflux. Studies with peptide and nonpeptide inhibitors of proteolysis suggested that active catalytic activity of trypsin was necessary for the inhibitory effect to be expressed. The degree of inhibition for both cholesterol exchange and mass efflux was dependent in a quantitatively similar manner on the time of incubation and the concentration of trypsin, suggesting that the mechanism of cholesterol exchange and mass efflux were similar at the level of the plasma membrane. Two other serine-proteases, thrombin and elastase, were also capable of inhibiting cholesterol removal in a similar manner. No cell death was observed by altered morphology, detachment, changes in DNA or protein content, or trypan blue exclusion even under the most severe proteolytic conditions. These studies suggest that protease-sensitive plasma membrane proteins play a role in cholesterol efflux in macrophages.

Pigeon aortic smooth muscle cells lack a functional low density lipoprotein receptor pathway.

The low density lipoprotein (LDL) receptor pathway was studied in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons and compared with rhesus monkey cells whose LDL receptor pathway has been previously characterized. Pigeon LDL was bound with high affinity in a saturable manner to both pigeon and monkey aortic smooth muscle cells. The kinetics of binding were different, however. LDL binding to pigeon cells exhibited positive cooperativity at low LDL concentrations and at least two classes of binding sites. The same pigeon LDL bound to monkey cells in a manner consistent with a single class of binding sites. Thus, these differences were a property of the pigeon cells and not the result of differences in the LDL. On the average, pigeon cells bound less than 50% the amount of LDL as monkey cells. Despite the surface binding to pigeon cells, little of the LDL was internalized, whereas pigeon LDL was actively internalized by monkey cells. Consistent with this observation, chloroquine and leupeptin had no effect on accumulation of LDL or on LDL degradation by pigeon cells, and incubation of pigeon cells with LDL produced no increase in cellular cholesteryl ester content. Binding of LDL to pigeon cells also differed from that of monkey cells by being unaffected by pretreatment with the proteolytic enzyme pronase, and by not requiring calcium. Binding was not specific for LDL since acetyl-LDL, and to a lesser degree HDL, were able to compete for LDL binding. Incubation with lipoprotein-deficient serum decreased LDL binding in pigeon cells while 25-OH cholesterol caused an increase in binding; both effects are opposite of that seen with the same LDL in mammalian cells. Preincubation with LDL or cholesterol dissolved in ethanol were without effect on LDL binding in pigeon cells, even though they produced significant increases in cellular free cholesterol content. In spite of the failure to internalize LDL, there was considerable degradation of LDL. This apparently occurred on the cell surface rather than by internalization and degradation within the lysosomes as occurs in mammalian cells. The functional significance of LDL binding to pigeon smooth muscle cells is unclear. The characteristics of binding resemble that of a nonspecific lipoprotein receptor referred to by others as the "lipoprotein receptor" or the "EDTA-insensitive receptor." It is apparent, however, that White Carneau pigeon aortic smooth muscle cells lack a functional LDL receptor pathway and in this way resemble cells from human beings with homozygous familial hypercholesterolemia or from Watanabe rabbits.

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway.

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.