Differences in adipocyte long chain fatty acid uptake in Osborne-Mendel and S5B/Pl rats in response to high-fat diets.

OBJECTIVE: To determine whether strain differences in adipocyte uptake of long chain fatty acids (LCFAs) contribute to differences in weight gain by Osborne-Mendel (OM) and S5B/Pl rats (S) fed a high-fat diet (HFD). SUBJECTS: Ninety-four adult (12-14-week old) male OM and S rats. MEASUREMENTS: Body weight; epididymal fat pad weight; adipocyte size, number, LCFA uptake kinetics; and plasma insulin and leptin during administration of HFD or chow diets (CDs). RESULTS: In both strains, rate of weight gain (RWG) was greater on an HFD than a CD; RWG on an HFD was greater, overall, in OM than S. A significant RWG increase occurred on days 1 and 2 in both strains. It was normalized in S by days 6-9 but persisted at least till day 14 in OM. RWGs were significantly correlated (P<0.001) with the V(max) for saturable adipocyte LCFA uptake (V(max)). In S, an increase in V(max) on day 1 returned to baseline by day 7 and was correlated with both plasma insulin and leptin levels throughout. In OM, a greater increase in V(max) was evident by day 2, and persisted for at least 14 days, during which both insulin and leptin levels remained elevated. Growth in epididymal fat pads on the HFD correlated with body weight, reflecting hypertrophy in OM and both hypertrophy and hyperplasia in S. CONCLUSIONS: (a) Changes in V(max) contribute significantly to changes in RWG on HFDs. (b) There are important strain differences in circulating insulin and leptin responses to an HFD. (c) Both insulin and leptin responses to an HFD are closely correlated with V(max) of adipocyte fatty acid uptake in S animals, but suggest early onset of insulin resistance in OM. Thus, differences in hormonal regulation of adipocyte LCFA uptake may underlie the different responses of OM and S to HFD.

Mitochondrial aspartate aminotransferase: direction of a single protein with two distinct functions to two subcellular sites does not require alternative splicing of the mRNA.

During differentiation of mouse 3T3-L1 fibroblasts to an adipocyte phenotype, the mitochondrial isoform of aspartate aminotransferase accumulates on the plasma membrane. The determination of whether this reflects translation of an alternatively spliced message lacking the mitochondrial leader sequence required cloning of the enzyme's uncommon a allele, for which these cells are homozygous. The 1.4-kb cDNA sequence of the a allele was obtained from oligo-dT-primed reverse-transcriptase PCR products amplified from FVB mouse RNA. It differed from the b allele at only 2 bp and one amino acid. By contrast, gene-specific primers generated an additional 1.4-kb fragment that differed from the b allele by approximately 1% of nucleotides, encoding four amino acid substitutions. This sequence proved to represent a recently diverged processed pseudogene. The presence of such pseudogenes can complicate interpretation of expressed-sequence-tag data and single-nucleotide-polymorphism genotyping studies. Using probes derived from the a allele, RNase protection analyses indicated that only a single message for the enzyme was present in 3T3-L1 fibroblasts and adipocytes, despite differences in subcellular protein distribution.

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.

Uptake of aluminum and gallium into tissues of the rat: influence of antibody against the transferrin receptor.

Transport of aluminum and gallium from blood into rat tissues following continuous i.v. infusion of metals in different chemical forms has been investigated. Tissue uptake of aluminum and gallium was similar and highly dependent on the chemical species of the metals. Aluminum and gallium accumulated in liver and spleen when infused in the chloride form. Raised citrate markedly enhanced aluminum and gallium uptake into renal cortex and bone; in contrast with gallium-transferrin, citrate increased uptake of 67Ga into renal cortex and bone by 8- and 14-fold respectively. Uptake of 67Ga with citrate into renal cortex was around 3 times smaller than that of aluminum. The antitransferrin receptor antibody OX-26 enhanced 67Ga uptake from gallium citrate into all rat tissues. 67Ga from purified gallium-transferrin was also taken into all tissues in the presence of OX-26, the effect being greatest in renal cortex and bone. No influence of antibody on aluminum transport into rat tissues was, however, observed when aluminum was infused in the citrate form. Therefore, transport of aluminum and gallium into tissues is not similar under all conditions. Transport of each metal occurs into all tissues in the presence of antitransferrin receptor antibody. The potential for such transport is much greater in the case of gallium. Transport of aluminum and gallium citrate complexes appears important especially in the renal cortex and bone.

Uptake of Ga-67 into rat cerebral hemisphere and cerebellum. Comparison with Fe-59.

Transferrin and transferrin receptors play an important role in the transport of iron into the brain. To determine whether gallium enters the brain by the same mechanism, uptakes of 67Ga and 59Fe have been compared under controlled conditions. Rates of gallium penetration into brain (K(in)) were four times slower than those for 59Fe. K(in) for 67Ga when infused with citrate were 0.88 +/- 0.24 and 0.94 +/- 0.39 x 10(-3) ml g-1h-1 for cerebral hemisphere and cerebellum, respectively. When infused as the transferrin complex, 67Ga uptake into the brain was not different from that when infused with citrate. The presence of the anti-transferrin receptor antibody OX-26 significantly reduced uptake of 59Fe by 60% and 64% into cerebral hemisphere and cerebellum, respectively. By contrast, pretreatment of rats with OX-26 enhanced the uptake of 67Ga into brain, particularly when infused with citrate; mean increases in uptake of 67Ga were 120% and 144% for cerebral hemisphere and cerebellum, respectively. Purified 67Ga-transferrin was also taken up into both brain regions examined in the presence of OX-26. These results indicate that the transport of non-transferrin bound gallium is an important mechanism for gallium uptake into brain.

Transport of iron in the blood-brain-cerebrospinal fluid system.

Iron is an important constituent in brain and, in certain regions, e.g., the basal nuclei, reaches concentrations equivalent to those in liver. It has a role in electron transfer and is a cofactor for certain enzymes, including those involved in catecholamine and myelin synthesis. Iron in CSF is likely to be representative of that in interstitial fluid of brain. Transferrin in CSF is fully saturated, and the excess iron may be loosely bound as Fe(II). Brain iron is regulated in iron depletion, suggesting a role for the blood-brain barrier (BBB). Iron crosses the luminal membrane of the capillary endothelium by receptor-mediated endocytosis of ferric transferrin. This results in an initial linear uptake of radioactive iron into brain at an average rate relative to serum of about 3.3 x 10(-3) ml x g of brain(-1) x h(-1) in the adult rat. This corresponds to about 80 nmol x kg(-1) x h(-1). Much higher rates occur in the postnatal rat. These increase during the first 15 days of life and decline thereafter. Within the endothelium, most of the iron is separated from transferrin, presumably by the general mechanism of acidification within the endosome. Iron appears to be absorbed from the vesicular system into cytoplasm and transported across the abluminal plasma membrane into interstitial fluid as one or more species of low molecular weight. There is some evidence that ionic Fe(II) is involved. Certainly Fe(II) ions presented on the luminal side rapidly cross the complete BBB, i.e., luminal and abluminal membranes. Within interstitial fluid, transported iron will bind with any unsaturated transferrin synthesized or transported into the brain-CSF system. Oligodendrocytes are one site of synthesis. From interstitial fluid, ferric transferrin is taken up by neurones and glial cells by the usual receptor-mediated endocytosis. Calculations of the amount of iron leaving the system with the bulk flow of CSF indicate that most iron entering brain across the capillary endothelium finally leaves the system with the bulk outflow of CSF through arachnoid villi and other channels. A system in which influx of iron into brain is by regulated receptor-mediated transport and in which efflux is by bulk flow is ideal for homeostasis of brain iron.

Uptake of 26-Al and 67-Ga into brain and other tissues of normal and hypotransferrinaemic mice.

Aluminium uptake from blood into tissues of control and homozygous hypotransferrinaemic (hpx/hpx) mice, following continuous intravenous infusion of 26Al and 67Ga, has been compared with that of gallium, a proposed tracer for aluminium. 26Al uptake into tissues of control (hpx/+ and +/+) mice occurred in the order (expressed as a space): bone 464.7 ml 100 g-1; renal cortex 102.9 ml 100 g-1; liver 13.0 ml 100 g-1; spleen 8.4 ml 100 g-1 and brain 0.8 ml 100 g-1. 67Ga uptakes were similar in liver, spleen and brain, but smaller in the renal cortex and bone, at one-third and one-fifth of the values for 26Al, respectively. In the hypotransferrinaemic mice, uptake of 67Ga into all tissues was increased, especially in renal cortex (ninefold) and bone (twentyfold) as compared with the controls. Increases in 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem of the hypotransferrinaemic mice were 3.8, 4.2 and 2.8 fold, respectively. 26Al uptake into tissues of the hypotransferrinaemic mice was similar to control values except in bone where it was three times greater. Pre-treatment of control animals with the anti-transferrin receptor antibody, RI7 208, enhanced 67Ga uptake in all tissues, the effect being greatest in renal cortex (tenfold) and bone (ninefold). 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem in the mice pre-treated with RI7 208 were 6.4, 6 and 10 times greater than in untreated mice, respectively. No influence of antibody on 26AI uptake into mouse tissues was observed except in spleen where it was three times greater than in untreated mice. Hence, transport of aluminium and gallium into mouse tissues is not similar under all conditions. Non-transferrin mediated transport of each metal can occur into all tissues, especially in renal cortex and bone, where gallium may be a suitable marker for aluminium.

Molecular factors influencing drug transfer across the blood-brain barrier.

A recently reported approach to the prediction of blood-brain drug distribution uses the general linear free energy equation to correlate equilibrium blood-brain solute distributions (logBB) with five solute descriptors: R2 an excess molar refraction term; pi2H, solute dipolarity or polarizability; alpha2H and beta2H, the hydrogen bond acidity or basicity, and Vx, the solute McGowan volume. In this study we examine whether the model can be used to analyse kinetic transfer rates across the blood-brain barrier in the rat. The permeability (logPS) of the blood-brain barrier to a chemically diverse series of compounds was measured using a short duration vascular perfusion method. LogPS data were correlated with calculated solute descriptors, and octanol-water partition coefficients (logP(oct)) for comparison. It is shown that a general linear free energy equation can be constructed to predict and interpret logPS values. The utility of this model over other physicochemical descriptors for interpreting logPS and logBB values is discussed.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Contrasting uptakes of 59Fe into spleen, liver, kidney and some other soft tissues in normal and hypotransferrinaemic mice. Influence of an antibody against the transferrin receptor.

Uptake of iron-59 from blood into various soft tissues of anaesthetized mice was investigated by continuous intravenous infusion of the radiotracer during 2 hr. The 59Fe was given either as ferrous chloride with ascorbate or as 59Fe-transferrin. Infusions were made into adult mice with and without pretreatment with a monoclonal antibody against transferrin receptors, and into hypotransferrinaemic mice and appropriate controls. In normal mice, 59Fe uptake into spleen was much higher than into other tissues and was 94-96% inhibited by the antibody. Inhibitions due to the antibody were less complete in liver and renal cortex, and there was evidence of some non-transferrin-mediated transport during infusion of 59Fe/ascorbate. In the hypotransferrinaemic mice, tissue uptakes of 59Fe during infusion of 59Fe/ascorbate were enormous, being two to three orders of magnitude greater than in the normal controls. The rank order for size of uptake was liver > renal cortex > pancreas > spleen > other tissues. All tissues examined have a considerable potential capacity for uptake of non-transferrin-bound iron, this being greatest in liver and renal cortex.

Transport of zinc-65 at the blood-brain barrier during short cerebrovascular perfusion in the rat: its enhancement by histidine.

Zinc-65 transport into different regions of rat brain has been measured during short vascular perfusion of one cerebral hemisphere with an oxygenated HEPES-containing physiological saline at pH 7.40. The [Zn2+] was buffered with either bovine serum albumin or histidine. In each case uptake was linear with time up to 90 s. 65Zn flux into brain in the presence of albumin followed Michaelis-Menten kinetics and for parietal cortex had a Km of 16 nM and a Vmax of 44 nmol/kg/min. Increasing concentrations of L-histidine enhanced 65Zn flux into brain at [Zn2+] values between 1 and 1,000 nM. The combined effect of [histidine] and [Zn2+] was best accounted for by a function of [ZnHis+], i.e., flux = 64.4.[ZnHis+]/(390 + [ZnHis+]) + 0.00378.[ZnHis+], with concentrations being nanomolar. D-Histidine had an influence similar to that of L-histidine. 65Zn flux in the presence of 100 microM L-histidine was not affected by either 500 microM L-arginine or 500 microM L-phenylanine. The results indicate specific transport of Zn2+ across the plasma membranes of brain endothelium. The enhancement due to histidine has been attributed to diffusion of ZnHis+ across unstirred layers "ferrying" zinc to and from transport sites.

Transport into retina measured by short vascular perfusion in the rat.

1. The short duration cerebrovascular perfusion method for measuring permeability of the blood-brain barrier has been adapted to measuring transport into the retina. 2. The method has been characterized on the one hand by comparing uptakes of radiotracers during HCO3(-)-buffered saline perfusion with those occurring after intravenous bolus injection of radioisotopes, and on the other by comparing uptake into retina with the uptake into frontal cerebral cortex. The mean permeability-surface area (PS) products (ml s-1 g-1) for [14C]urea and [14C]thiourea in the perfused retina were 1.2 +/- 0.26 x 10(-3) and 2.1 +/- 0.01 x 10(-3) respectively. The intravenous injection method gave comparable values for [14C]urea and [14C]thiourea of 1.6 +/- 0.28 x 10(-3) and 3.24 +/- 0.55 x 10(-3). The rates of uptake of the hydrophilic solutes were 2- to 7-fold greater than in brain. 3. Retinal and choroidal capillary perfusion fluid flow rates were measured using a diffusible flow marker ([14C]diazepam) and a particulate indicator (15 microns cerium141-labelled microspheres). Results using both flow markers confirmed that both capillary networks supplying the retina were being adequately perfused.

Determination of aluminium in different tissues of the rat by atomic absorption spectrometry with electrothermal atomization.

Atomic absorption spectrometry with electrothermal atomization was used for the determination of aluminium in brain, liver, spleen, kidney cortex, skeletal muscle and bone of the rat following digestion by nitric acid and in serum following simple dilution and in situ oxygen ashing. The method of standard additions in the presence of a chemical modifier, ammonium dihydrogen-phosphate, was essential for bone tissues. The detection limits ranged from 3 to 58 ng per gram of wet mass of tissue and were 4-19 times lower than the observed physiological levels of aluminium. The between-day precision for serum was 8.9% at a mean concentration of 6.8 micrograms I-1 and 2.4% at a mean concentration of 125.3 micrograms I-1. Additionally, repeated analyses of National Institute of Standards and Technology Standard Reference Material 1577b Bovine Liver gave a relative standard deviation of 12.2% (mean concentration = 0.8 microgram g-1). Of the tissues studied, bone had at least ten times higher levels of aluminium than others (0.959 +/- 0.322 micrograms g-1). The aluminium concentration in cerebellum (0.073 +/- 0.043 micrograms g-1) was approximately twice that in the cerebral hemisphere (0.034 +/- 0.009 micrograms g-1).

Permeability of the blood-brain barrier to lead.

This review examines the kinetics and possible mechanisms of lead transport into brain across the microvessel endothelium (the blood-brain barrier). Although severe lead poisoning both in neonatal rats and in young children may cause microvessel damage, there is little evidence that there is either damage or even disturbance of specific transport mechanisms at blood leads < 80 micrograms/dl. When 203Pb was continuously infused intravenously into adult rats, radiotracer uptake into different brain regions was linear with time up to 4 hours, reaching spaces in relation to plasma of 6.6 - 8.2 ml/100 g in cerebral tissues at one hour. The concentration of free Pb+ in serum is of the order of 10(-12)M, the majority of lead being bound to protein and to sulfhydryl compounds, such as L-cysteine. Transport into brain has been further studied during short vascular perfusion of one cerebral hemisphere of the rat with oxygenated and buffered physiological saline. This allows total control of the fluid perfusing the cerebral microvessels. In the absence of organic ligands for lead, 203Pb entered brain very fast, with a space of 9.7 ml/100 g in frontal cortex at one min. The presence of albumin, L-cysteine or EDTA abolished measurable uptake. Experiments designed to reveal a role for the anion exchanger or calcium channels gave negative results. However, the effects of potassium depolarization and of varying pH indicated that the lead species passively entering the endothelium might be PbOH+. Experiments with various metabolic inhibitors, including vanadate, suggested that Pb uptake in the endothelium is mitigated by active back transport of lead into blood by the Ca-ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Rate of 59Fe uptake into brain and cerebrospinal fluid and the influence thereon of antibodies against the transferrin receptor.

Uptake of 59Fe from blood into brains of anaesthetized rats and mice has been studied by intravenous infusion of [59Fe]ferrous ascorbate or of 59Fe-transferrin, the results not being significantly different. Uptakes in the rat were linear with time, but increased at longer times in the mouse. Transfer constants, K(in) (in ml/g/h x 10(3)), for cerebral hemispheres were 5.2 in the adult rat and 5.6 in the mouse. These K(in) values corresponded to 59Fe influxes of 145 and 322 pmol/g/h, respectively. 59Fe uptake into the mouse brain occurred in the following order: cerebellum > brainstem > frontal cerebral cortex > parietal cortex > occipital cortex > hippocampus > caudate nucleus. In genetically hypotransferrinaemic mice, 59Fe uptake into brain was 80-95 times greater than in To strain mice. Pretreatment of young rats and mice with monoclonal antibodies to transferrin receptors, i.e., the anti-rat immunoglobulin G OX 26 and the anti-mouse immunoglobulin M RI7 208, inhibited 59Fe uptake into spleen by 94% and 98%, respectively, indicating saturation of receptors. The antibodies reduced 59Fe uptake into rat brain by 35-60% and that into mouse brain by 65-85%. Although a major portion of iron transport across the blood-brain barrier is normally transferrin-mediated, non-transferrin-bound iron readily crosses it at low serum transferrin levels.

Observations on the chemical nature of lead in human blood serum.

1. The binding of lead to human blood serum, and components of serum, was studied by titration with the addition of Pb(NO3)2 solution, monitoring the free Pb2+ concentration with a Pb2+ electrode, and by equilibrium dialysis. 2. In fresh serum, about 4999 out of 5000 parts of added lead were bound. This suggests that the free Pb2+ concentration is around 1/5000th of the total lead concentration in the serum of normal subjects, i.e. about 1 x 10(-12) mol/l. 3. About 60% of the binding of lead in serum is abolished by standing in air, by dialysis or by treatment with N-ethylmaleimide. This appears to be due to the presence of thiol compounds, mainly cysteine. The remaining 40% appears to be due to protein, mainly albumin.