Functional compensation for adipose differentiation-related protein (ADFP) by Tip47 in an ADFP null embryonic cell line.

Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.

Kinetics of 14C distribution after tracer dose of 14C-lutein in an adult woman.

Lutein is an oxygenated carotenoid (xanthophyll) found in dark green leafy vegetables. High intakes of lutein may lower the risk of age-related macular degeneration. Current understanding of human lutein metabolism as it might occur in vivo is incomplete. Therefore, we conducted a feasibility study where we dosed a normal adult woman with 14C-lutein (125 nmol, 36 nCi 14C), dissolved in olive oil (0.5 g/kg body weight) and mixed in a banana shake. Blood, urine, and feces collected before the dose was administered served to establish baseline values. Thereafter, blood was collected for 63 d following the dose, while feces and urine were collected for 2 wk post-dose. The 14C contents in plasma, urine, and feces were measured by accelerator MS. The 14C first appeared in plasma 1 h after dosing and reached its highest level, approximately2.08% of dose/L plasma, at 14 h post-dose. The plasma pattern of 14C did not include a chylomicrons/VLDL (intestinal) peak like that when the same subject received 14C-beta-carotene (a previous test), suggesting that lutein was handled differently from beta-carotene by plasma lipoproteins. Lutein had an elimination half-life (t1/2) of approximately10 d. Forty-five percent of the dose of 14C was eliminated in feces and 10% in urine in the first 2 d after dosing. Quantifying human lutein metabolism is a fertile area for future research.

Structure of a lipid droplet protein; the PAT family member TIP47.

The perilipin/ADRP/TIP47 (PAT) proteins localize to the surface of intracellular neutral lipid droplets. Perilipin is essential for lipid storage and hormone regulated lipolysis in adipocytes, and perilipin null mice exhibit a dramatic reduction in adipocyte lipid stores. A significant fraction of the approximately 200 amino acid N-terminal region of the PAT proteins consists of 11-mer helical repeats that are also found in apolipoproteins and other lipid-associated proteins. The C-terminal 60% of TIP47, a representative PAT protein, comprises a monomeric and independently folded unit. The crystal structure of the C-terminal portion of TIP47 was determined and refined at 2.8 A resolution. The structure consists of an alpha/beta domain of novel topology and a four-helix bundle resembling the LDL receptor binding domain of apolipoprotein E. The structure suggests an analogy between PAT proteins and apolipoproteins in which helical repeats interact with lipid while the ordered C-terminal region is involved in protein:protein interactions.

Palatability affects the percentage of metabolizable energy as protein selected by adult beagles.

The percentage of protein that dogs voluntarily choose and the effect of palatability on the quantity selected were determined. Six beagles were offered a choice of two isoenergetic purified diets containing 0 vs. 25, 9 vs. 32, 18 vs. 32, 18 vs. 48 and 25 vs. 48% metabolizable energy from protein (MEp). To examine whether palatability modifies the choice, the dogs were offered 0 vs. 25% MEp, with the 0% protein diet containing 2.9 times more sucrose than the diet containing 25% MEp. To determine the effect of concentration of protein in the diet on dietary choice and plasma amino acid concentrations (PAA), dogs were adapted to 9% MEp, followed by a choice of diets containing 9 vs. 32% MEp. The choice was repeated after adaptation to a diet containing 32% MEp. Dogs selected diets to obtain 21-27% of the MEp (mean, 25% MEp; median, 27% MEp) when sucrose was kept at 6.4%. When the protein-free diet contained 25% sucrose, dogs selected 17% of MEp, but increased food intake to ingest about the same amount of protein per day. PAA did not correlate linearly with protein intake. Food intake and total PAA were the lowest after consumption of the 9% MEp diet. We conclude that when fed equally bland diets, dogs select food to ingest approximately 25% MEp. As a palatability enhancer, sucrose increases food intake and selection of the diet containing the higher sucrose concentration.

Dual isotope test for assessing beta-carotene cleavage to vitamin A in humans.

BACKGROUND: The ability of beta-carotene to deliver bioactive retinoids to tissues is highly variable. A clearer understanding of the environmental and genetic factors that modulate the vitamin A potential of beta-carotene is needed. AIM OF STUDY: Assess the vitamin A value of orally administered beta-carotene relative to a co-administered reference dose of preformed vitamin A. METHODS: Equimolar doses (30 micromol) of hexadeuterated D6 beta-carotene and D6 retinyl acetate were orally co-administered in an emulsified formulation to a male subject. The plasma concentration time courses of D6 retinol (derived from D6 retinyl acetate) and bioderived D3 retinol (from D(6) beta-carotene) were determined for 554 h postdosing using gas chromatography/mass spectrometry. Intact D6 beta-carotene plasma concentrations were determined by high-pressure liquid chromatography. The ratio of the two forms of vitamin A, D6 retinol/D3 retinol, at any single time point is postulated to reflect the quantity of vitamin A derived from beta-carotene relative to preformed vitamin A. Additionally, a minute amount of 14C beta-carotene (50 nCi; 0.27 microg) was included in the oral dose and cumulative 24-h stool and urine samples were collected for two weeks to follow absorption and excretion of the b-carotene. The 14C nuclide was detected using accelerator mass spectrometry (AMS). Results During the absorption/distribution phase (3-11 h) the D6/D3 ratio of the two retinols was not stable and ranged between a value of 3 and 16. Between 11 and 98 h postdosing the ratio was relatively stable with a mean value of 8.5 (95 % CI: 7.5, 8.7). These data suggest that in this subject and under these conditions, 8.5 moles of beta-carotene would provide a vitamin A quantity equivalent to 1 mole of preformed vitamin A. On a mass basis, 15.9 microg of beta-carotene was equivalent to 1 microg of retinol. The total administered beta-carotene was found to be 55 % absorbed by AMS analysis of cumulative stool. CONCLUSION: The co-administration of D6 beta-carotene and D6 retinyl acetate provides a technique for assessing individual ability to process beta-carotene to vitamin A. The results indicate that a single time point taken between 11-98 h after dose administration may provide a reliable value for the relative ratio of the two forms of vitamin A. However, results from more subjects are needed to assess the general utility of this method.

Variability in conversion of beta-carotene to vitamin A in men as measured by using a double-tracer study design.

BACKGROUND: The vitamin A activity of beta-carotene is variable and surprisingly low in women. The reasons for this are not well understood. The vitamin A activity of beta-carotene in men is still uncertain. Contributions of dietary factors compared with individual traits are largely unknown. OBJECTIVE: Our objective was to measure the intrinsic variability in the vitamin A activity of beta-carotene among healthy, well-fed men living in a controlled environment. DESIGN: We used a double-tracer test-retest design. We dosed 11 healthy men orally with 30 micromol hexadeuterated (D6) retinyl acetate (all-trans-19,19,19,20,20,20-[2H6]retinyl acetate) and then with 37 micromol D6 beta-carotene (19,19,19,19',19',19'-[2H6]beta-carotene) 1 wk later. Doses were taken with breakfasts containing 16 g fat. We measured D6 retinol, D6 beta-carotene, and trideuterated (D3) retinol (derived from D6 beta-carotene) concentrations in plasma. Areas under the plasma concentration x time since dosing curves (AUCs) were determined for D6 retinol, D6 beta-carotene, and D3 retinol. RESULTS: All men had detectable D6 retinol concentrations in plasma. The mean (+/-SE) absorption of D6 beta-carotene in all subjects was 2.235 +/- 0.925%, and the mean conversion ratio was 0.0296 +/- 0.0108 mol retinol to 1 mol beta-carotene. Only 6 of 11 men had sufficient plasma concentrations of D6 beta-carotene and D3 retinol that we could measure. The mean absorption of D6 beta-carotene in these 6 subjects was 4.097 +/- 1.208%, and the mean conversion ratio was 0.0540 +/- 0.0128 mol retinol to 1 mol beta-carotene. CONCLUSION: The vitamin A activity of beta-carotene, even when measured under controlled conditions, can be surprisingly low and variable.