Model-based compartmental analysis indicates a reduced mobilization of hepatic vitamin A during inflammation in rats

Vitamin A (VA) kinetics was studied in rats with marginal VA stores before, during, and after inflammation. Rats received orally [11,12-(3)H(N)]retinol ([(3)H]VA; day 0), and inflammation was induced on day 21 with lipopolysacchride (LPS) for 3 days (n = 5) or recombinant human interleukin-6 (rhIL-6) for 7 days (n = 5). Both the fraction of [(3)H]VA and retinol concentrations in plasma were reduced significantly by LPS or rhIL-6. Compartmental analysis using the Windows version of Simulation, Analysis, and Modeling software was applied to group mean data, and non-steady-state models were developed. After absorption, VA kinetics was described by a three-compartment model that included plasma, kidney/interstitium, and liver/carcass. Four mechanisms decreasing plasma retinol were investigated: increased urinary excretion, increased irreversible loss, increased movement into interstitium, and decreased hepatic mobilization. Modeling demonstrated that a 79% reduction in hepatic mobilization of retinol (from 4.3 to 0.9 nmol/h) by 15 h after LPS best accounted for the observed changes in plasma VA kinetics (sum of squares = 9.05 x 10(-07)). rhIL-6 caused an earlier reduction (75% by 5.6 h). These models predicted a return to control values by 10 days after inflammation. If prolonged, inflammation-induced hyporetinolemia can render hepatic retinol unavailable to extrahepatic tissues, possibly leading to their impaired function, as observed in VA-deficient children with measles infection.     

Study on the mechanism of interference of 3,4,3',4'-tetrachlorobiphenyl with the plasma retinol-binding proteins in rodents

The mechanism of plasma retinol reduction in rodents by 3,4,3',4'-tetrachlorobiphenyl (TCB) was investigated by radioimmunochemical analysis of the amounts of circulating and hepatic retinol-binding protein (RBP) and transthyretin (TTR) in exposed and control animals. Plasma RBP concentrations were markedly reduced in C57BL/Rij mice (50%) at 4 days, in DBA/2 mice (37-41%) at 4 and 8 days, and in Sprague-Dawley rats (58%) at 2 days after exposure to TCB. These reductions paralleled the time course of reduction of plasma retinol after exposure to TCB. Hepatic RBP concentrations were somewhat increased in TCB-treated animals, especially in the C57BL/Rij mouse and Sprague-Dawley rat. However, the release of hepatic RBP into the circulation was not blocked by TCB treatment, as analysed in vitamin A deficient rats. In addition, the amount of plasma TTR was in the normal range in TCB-treated rats. The dissociation constants of the RBP-TTR complex as analysed by polarization of fluorescence appeared to be significantly increased (from 0.5 x 10(-7) M-1 to 2.4 x 10(-7) M-1) in the presence of a TCB metabolite, isolated from plasma of TCB-treated rats. In addition, the estimated number of binding sites for RBP on the TTR molecule was reduced (from 2.8 to 1.7 sites) upon treatment of TTR with the TCB metabolite. These data support the hypothesis that plasma retinol reduction by TCB might result from a weakening of the RBP-TTR complex, in the presence of the TCB metabolite bound to the TTR.     

Retinol-binding protein synthesis and secretion by the rat visceral yolk sac. Effect of retinol status

Studies were conducted to explore in rats the role of retinol in the regulation of the synthesis and secretion of retinol-binding protein (RBP) by the visceral yolk sac compared to the liver. Previous studies have shown that in retinol deficiency, hepatic RBP secretion is specifically inhibited, whereas hepatic RBP synthesis rate is unchanged. Retinol-depleted, retinoic acid-supplemented female rats were mated, and maternal liver, fetal liver, and visceral yolk sac were obtained at 14 days of gestation (retinol-depleted group). A group of identically treated, retinol-depleted rats were repleted with retinol on the 14th day of gestation, and the same tissues were collected 6 h later (retinol-repleted group). Normal female rats were used as controls. RBP was assayed by radioimmunoassay and RBP mRNA levels by RNase protection assay using a rat RBP cDNA clone. RBP levels in the visceral yolk sac were elevated 10-fold in the retinol-depleted as compared to the control rats and had declined to near normal values in the retinol-repleted animals. The relative levels of RBP mRNA in the visceral yolk sac were very similar in all three groups of rats. Thus, as in the liver, in the visceral yolk sac retinol deficiency inhibits RBP secretion without altering RBP mRNA levels. In the visceral yolk sac, as in the liver, retinol status appears to regulate RBP secretion specifically, without affecting the rate of RBP biosynthesis.     

Retinol kinetics in unsupplemented and vitamin A-retinoic acid supplemented neonatal rats: a preliminary model

Vitamin A (VA) metabolism in neonates is virtually uncharacterized. Our objective was to develop a compartmental model of VA metabolism in unsupplemented and VA-supplemented neonatal rats. On postnatal day 4, pups (n = 3/time) received 11,12-[³H]retinol orally, in either oil (control) or VA combined with retinoic acid (VARA) [VA (∼6 mg/kg body weight) + 10% retinoic acid]. Plasma and tissues were collected at 14 time points up to 14 days after dose administration. VARA supplementation rapidly, but transiently, increased total retinol mass in plasma, liver, and lung. It decreased the peak fraction of the dose in plasma. A multi-compartmental model developed to fit plasma [³H]retinol data predicted more extensive recycling of retinol between plasma and tissues in neonates compared with that reported in adults (144 vs. 12–13 times). In VARA pups, the recycling number for retinol between plasma and tissues (100 times) and the time that retinol spent in plasma were both lower compared with controls; VARA also stimulated the uptake of plasma VA into extravascular tissues. A VARA perturbation model indicated that the effect of VARA in stimulating VA uptake into tissues in neonates is both dramatic and transient.     

Changes in vitamin A status following prolonged immobilization (simulated weightlessness).

A study was conducted to investigate the effects of a simulated weightlessness induced by chronic immobilization on vitamin A status. To simulate the stress condition of weightlessness, rats were suspended for 10 days in a special jacket to which metal chains were attached. Animals received a commercial stock diet. Control rats were pair-fed in reference to the suspended rats. As compared with the control, prolonged immobilization resulted in a decrease in body weight gain and an increase in adrenal weight occurred. In the suspended rats, serum concentrations of retinol and retinol-binding protein (RBP) declined. Hepatic retinyl palmitate content increased, and the hepatic retinol level was decreased. The prolonged immobilization led to significantly reduced retinyl palmitate levels in the testis and lung as well as lowered testicular retinol levels. The results suggest that the stress state induced by prolonged immobilization caused accumulation of hepatic retinyl palmitate, decreasing the serum retinol concentration and retinyl ester content in the extrahepatic tissues.     

Effect of hexachlorobiphenyl on vitamin A homeostasis in the rat

Vitamin A status and turnover were examined in rats that had been exposed to chronic dietary treatment of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 1 mg/kg diet. HCB caused hepatic depletion and renal accumulation of vitamin A, and a 1.7-fold increase in the serum retinol concentration. Intravenously administered [3H]retinol bound to retinol binding protein-transthyretin complex (RBP-TTR complex) was used to study the dynamics of circulatory retinol in these rats. In HCB-treated rats, the plasma turnover rate of retinol was increased compared to vitamin A-adequate untreated controls. HCB caused a 50% reduction of total radioactivity in liver, and, except for 0.5 h after the [3H]retinol-RBP-TTR dose, the specific activity of the hepatic retinyl ester pool was greater compared to control rats. The kidneys of HCB-treated rats accumulated radioactivity in the retinyl ester fraction. HCB also caused a 50% reduction in adrenal radioactivity compared with control rats. Urinary and fecal excretion of radioactivity was 3-fold higher in HCB-treated rats as compared to controls. Our findings demonstrate that chronic HCB feeding results in expansion of plasma vitamin A mass, in changes of liver and kidney retinol and retinyl ester pool dynamics and in an increased metabolism of vitamin A.     

Compartmental modeling of whole-body vitamin A kinetics in unsupplemented and vitamin A-retinoic acid-supplemented neonatal rats

Little is known about the contribution of different tissues to whole-body vitamin A (VA) kinetics in neonates. Here, we have used model-based compartmental analysis of tissue tracer kinetic data from unsupplemented (control) and VA-retinoic acid (VARA)-supplemented neonatal rats to determine VA kinetics in specific tissues under control and supplemented conditions. First, compartmental models for retinol kinetics were developed for individual tissues, and then an integrated compartmental model incorporating all tissues was developed for both groups. The models predicted that 52% of chylomicron (CM) retinyl ester was cleared by liver in control pups versus 22% in VARA-treated pups, whereas about 51% of VA was predicted to be extrahepatic in 4- to 6-day-old unsupplemented neonatal rats. VARA increased CM retinyl ester uptake by lung, carcass, and intestine; decreased the release into plasma of retinol that had been cleared by liver and lung as CM retinyl esters; stimulated the uptake of retinol from plasma holo-retinol binding protein into carcass; and decreased the retinol turnover out of the liver. Overall, neonatal VA trafficking differed from that previously described for adult animals, with a larger contribution of extrahepatic tissues to CM clearance, especially after VA supplementation, and a significant amount of VA distributed in extrahepatic tissues.     

Effect of retinoic acid and apo-RBP on serum retinol concentration in acute renal failure

We recently demonstrated a rapid up-regulation of serum retinol-retinol binding protein-transthyretin concentration in rats with short-term acute renal failure. We examine the effect of retinoic acid and apo-retinol binding protein (apo-RBP) on the up-regulation of serum retinol in renal failure. Injection of retinoic acid (10 micrograms) into rats with acute renal failure or sham-operated rats increased circulatory retinoic acid concentration 29-fold within 2 h but did not influence serum retinol concentration in either group. Injection of a large dose of retinoic acid (100 micrograms) decreased serum retinol concentration in rats with acute renal failure (19%) and sham-operated rats (29%). These results suggest that changes in serum retinoic acid concentration within the near-physiological range have no effect on regulation of hepatic retinol release. Injection of a large dose of retinoic acid may depress serum retinol indirectly via a retinol sparing effect in target tissues. In rats with renal failure the serum retinol concentration, elevated 44-52% above that of sham-operated controls, was also increased to 70-164% above controls by the injection of 52-63 micrograms of apo-RBP. This suggests that circulatory apo-RBP can up-regulate serum retinol. Circulatory apo-RBP may be a positive physiological feedback signal from peripheral tissues for hepatic release of retinol.     

Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in a synergistic manner in neonatal rats

Vitamin A (VA) is stored in tissues predominantly as retinyl esters (REs), which provide substrate for the production of bioactive retinoids. Retinoic acid (RA), a principal metabolite, has been shown to induce postnatal lung development. To better understand lung RE storage, we compared VA (given as retinyl palmitate), RA, and a nutrient-metabolite combination, VARA, given orally on postnatal days 5-7, for their ability to increase lung RE in neonatal rats. VARA increased lung RE significantly [ approximately 14, 2.4, 2.1, and <1 nmol/g for VARA, VA, RA, and control (C), respectively; P < 0.001]; the increase by VARA was more than additive compared with the effects of VA and RA alone. Lung histology and morphometry were unchanged. In a 6 h metabolic study, providing [(3)H]retinol with VARA, compared with VA or C, increased the uptake of newly absorbed (3)H by 3-fold, indicating that VARA stimulated the uptake of [(3)H]retinol and its retention as [(3)H]RE in neonatal lungs. After cessation of VARA, lung RE remained increased for 9 d afterward, through the period of alveolar development. In conclusion, VARA, a 10:1 nutrient-metabolite combination, increased lung RE significantly compared with VA alone and could be a promising therapeutic option for enhancing the delivery of VA to the lungs.     

Structure-assisted discovery of the first non-retinoid ligands for Retinol-Binding Protein 4

Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4–TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.     

Mechanism of the antiulcerogenic effect of IL-11 on acetic acid-induced gastric ulcer in rats

The purpose of this study was to evaluate the healing effect of interleukin-11 (IL-11) on acetic acid-induced gastric ulcer in rats. Gastric ulcers were induced in male Wistar rats by applying acetic acid to the fundus of the stomach. Recombinant human interleukin-11 (rhIL-11 100 microg/kg/twice daily, subcutaneously) was administered starting on the 2nd day before ulcer induction up through the 7th day after ulcer induction. Control rats were injected with bovine serum albumin. At 12 hours and 7 days after ulcer induction, the animals were sacrificed, and the ulcer index, proliferating cell nuclear antigen (PCNA) expression, and IL-11alpha receptor expression in the gastric tissues were studied. The ulcer index of the rhIL-11-treated rats was significantly lower than that of the control rats at the 7th day. The expression of PCNA as evaluated by Western blotting and immunohistochemistry, was enhanced in both the mucosal proliferative zone and proper muscle layer of the rhIL-11-treated rats in comparison with that in the control rats. IL-11alpha receptor expression was observed in the mucosal neck cells of the rhIL-11-treated rats and control rats. These findings suggest that IL-11 accelerates ulcer healing by inducing the proliferation of mucosal and muscular cells.     

Metabolism and secretion of retinol transport complex in acute renal failure

Tissue uptake and distribution of retinol from circulatory vitamin A transport complex was studied in order to determine the origin of the increased serum retinol in rats with short-term acute renal failure. In rats with acute renal failure, serum retinol increased 37-70% within 2 h after surgery. After an injection of donor plasma containing 1.8 muCi of [3H]retinol in retinol transport complex, in rats with renal failure the ability to clear radioactivity was decreased 36% by 0.5 h and 57% by 2 h, as compared to sham-operated rats. The uptake and distribution of radioactivity by nonrenal tissues was similar in rats with acute renal failure and with intact kidneys. The lack of renal function did not alter hepatic cycling of [3H]retinol from the circulation and thus could not account for the increased serum retinol in renal failure. When hepatic release of retinol-retinol binding protein was blocked by colchicine, the up-regulation of serum retinol, normally observed in rats with acute renal failure, was abolished. Our studies provide strong evidence that kidney has an important role in maintaining serum retinol homeostasis by influencing the release of retinol-retinol binding protein from liver into circulation. Peripheral tissue uptake of circulatory retinol and hepatic cycling of nonutilized retinol are not directly influenced by the kidney.     

Effects of dietary salt intake on plasma arginine

Because L-arginine is degraded by hepatic arginase to ornithine and urea and is transported by the regulated 2A cationic amino acid y(+) transporter (CAT2A), hepatic transport may regulate plasma arginine concentration. Groups of rats (n = 6) were fed a diet of either low salt (LS) or high salt (HS) for 7 days to test the hypothesis that dietary salt intake regulates plasma arginine concentration and renal nitric oxide (NO) generation by measuring plasma arginine and ornithine concentrations, renal NO excretion, and expression of hepatic CAT2A, and arginase. LS rats had lower excretion of NO metabolites and cGMP, lower plasma arginine concentration (LS: 83 +/- 7 vs. HS: 165 +/- 10 micromol/l, P < 0.001), but higher plasma ornithine concentration (LS: 82 +/- 6 vs. HS: 66 +/- 4 micromol/l, P < 0.05) and urea excretion. However, neither the in vitro hepatic arginase activity nor the mRNA for hepatic arginase I was different between groups. In contrast, LS rats had twice the abundance of mRNA for hepatic CAT2A (LS: 3.4 +/- 0.4 vs. HS: 1.6 +/- 0.5, P < 0.05). The reduced plasma arginine concentration with increased plasma ornithine concentration and urea excretion during LS indicates increased arginine metabolism by arginase. This cannot be ascribed to changes in hepatic arginase expression but may be a consequence of increased hepatic arginine uptake via CAT2A.     

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR—RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR—RBP concentrations determined by this method are highly correlated to holo-TTR—RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR—RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR—RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.     

Ultrastructural localization of plasma retinol-binding protein in rat liver

Immunocytochemical studies were carried out to examine the subcellular localization of plasma retinol-binding protein (RBP) in rat liver. The studies used normal, retinol-deficient, and retinol-repleted retinol-deficient rats with or without colchicine pretreatment. Affinity-purified monomeric Fab' fragments from the IgG fraction of rabbit anti-rat RBP were conjugated to horseradish peroxidase. This conjugate effectively penetrated into tissue sections and enabled RBP to be localized by high resolution immunoelectron microscopy. In the normal liver parenchymal cell, RBP was found to be localized in the synthetic and secretory structures including endoplasmic reticulum (ER), Golgi complex (GC), and secretory vesicles. With the method used, significant localization of RBP was not observed in hepatic cells other than parenchymal cells. The distribution of RBP-positive areas within parenchymal cells changed markedly with retinol depletion. Thus, a heavy accumulation of RBP in the ER, accompanied by a marked decrease of the RBP-positive GC and secretory vesicles, was demonstrated in liver parenchymal cells from retinol-deficient rats. After repletion of deficient rats with retinol, the RBP that accumulated in the ER appeared to move rapidly from the ER through GC and secretory vesicles to the cell surface. Pretreatment with colchicine led to marked increase in RBP-positive secretory vesicles in retinol-repleted rat liver parenchymal cells. The results reported here demonstrate that the specific block in hepatic RBP secretion seen in retinol deficiency involves an inhibition of the movement of RBP from the ER to the GC in the parenchymal cell.     

A multicompartmental model of vitamin A kinetics in rats with marginal liver vitamin A stores

A linear, first-order, constant-coefficient multicompartmental model is presented which describes the dynamics of [3H]retinol turnover in adult rats with normal plasma retinol concentrations but low liver stores (less than 100 micrograms of retinol equivalents). To fit plasma and tissue (liver, kidney, and rest of carcass) tracer and tracee data, eight physiological compartments were required in the model: two in plasma (proposed to correspond to the retinol transport complex, and retinyl esters in plasma lipoproteins) and two each in liver, kidneys, and other extrahepatic tissues. Extensive recycling of retinol among plasma, liver, and the rest of carcass was also required. The model predicted that 44% of whole body vitamin A (143 micrograms) was in extrahepatic tissues. The vitamin A utilization rate (system disposal rate) was 6.9 micrograms of retinol equivalents/day. The system residence time (mean sojourn time) for vitamin A was 21 days, and the fractional catabolic rate for the system was 5%/day. The mean transit time (turnover time) for vitamin A in its plasma retinol transport complex was 0.078 days (1.9 hr); the residence time was 0.98 day, versus 11 days in the liver, 9 days in carcass, and 0.54 days in kidneys. The model predicted that, of the plasma turnover, 48% recycled to the liver and 52% to extrahepatic tissues. The liver retinol secretion rate was 48 micrograms/day, more than half of which was from recycled plasma retinol. Since the plasma retinol turnover rate (87 micrograms/day) was 13 times the system disposal rate, the data suggest that this is a high response system in which changes in the dynamics of recycling of retinol allow for rapid adjustment in vitamin A distribution in response to changes in nutritional, metabolic, or physiological conditions; and in which plasma retinol levels are controlled homeokinetically by changes in hepatic and extrahepatic recycling of holo retinol-binding protein.     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.