Stimulation of glucose transport in rat thymocytes by human albumin preparations.

Attempts to disclose effects of fatty acids on glucose transport in rat thymocytes led us to observe that a fatty acid-free human albumin preparation (0.2%) stimulated glucose transport dramatically and quickly. Of the next several albumin preparations tested, a fatty acid-free (acid/charcoal extracted) human albumin preparation stimulated strongly, an unextracted human albumin preparation did not stimulate, a charcoalextracted bovine albumin preparation did not stimulate, and an unextracted bovine albumin preparation did not stimulate glucose transport. We suspected that acid charcoal extraction unmasked a stimulatory factor peculiar to human albumin. However, we have since found unextracted human albumin preparations with stimulatory activities comparable to those of extracted preparations. Lack of stimulation is the exception. Addition of fatty acids to fatty-acid free albumin did not reduce stimulatory activity, and charcoal extraction of an unstimulatory preparation of human albumin did not evoke stimulatory activity. The stimulatory activity was partially destroyed by trypsin treatment. We suspected contamination by insulin-like activity. We were unable to mimic the stimulatory effect with insulin, sulfation factor, multiplication-stimulating activity, T lymphocyte growth factor, or epidermal growth factor. In gel filtration and DEAE-sephadex chromatography, the stimulatory activity eluted with the albumin peak. Treatment with excess N-ethylmaleimide, 4,4’-dithiodipyridine, or benzoquinone did not obliterate activity. Treatment with 1 molecule of mercaptoethanol/lO molecules of albumin or 1 molecule of dithiothreitol/50 molecules of albumin did obliterate activity. From this we conclude that the stimulatory agent contains a highly reactive critical disulfide, is a minor component of the preparation, and is active at concentrations less than 3 l lo-’ M. The cellular response was not demonstrably Ca”-dependent and appeared to be slightly antagonized by catalase.


Attempts
to disclose effects of fatty acids on glucose transport in rat thymocytes led us to observe that a fatty acid-free human albumin preparation (0.2%) stimulated glucose transport dramatically and quickly. Of the next several albumin preparations tested, a fatty acid-free ( and is active at concentrations less than 3 l lo-' M. The cellular response was not demonstrably Ca"-dependent and appeared to be slightly antagonized by catalase.
In studies intended to test for possible effects of fatty acids on glucose transport in rat thymocytes, we found that a fatty acid-free albumin preparation stimulates glucose transport dramatically and quickly. We were curious about the effect and decided to characterize it further. In the present study, we report some properties of the effective agent and some features of the cellular response. We do not know whether this agent is one of the known serum factors such as a nonsuppressible insulin-like activity (l-3), a somatomedin (4-

Human Albumin Preparations Stimulate
Glucose Transport in Rat Thymocytes-As seen in control experiments of Fig. 1 and all other figures, ['4C]methylglucose exits the thymocytes in two phases. The fast phase has a half-time of about l-3 min (10,14) and is essentially finished by 10 min. The slow phase has a half-time of 30-50 min. We (15) and others (16) have shown that the two phases reflect transport of two kinds of cells, active and quiescent. About % of the methylglucose space is in active cells, and % are in quiescent cells. In these experiments, where cells were loaded with ['"Cl ' The abbreviation used is: 4-PDS, 4,4'-dithiodipyridine. Effects of various lots of human albumin on methylglucose efflux. Cells were loaded in normal medium. The albumin lots were present at 1% in the media with which the cells were diluted to initiate efflux. Lots 701-9310 and 110F-9350 were purchased from Sigma as fatty acid-free human albumin (A1887). Lot 30F-02271 (Sigma A2386) had not been treated for fatty acid removal. methylglucose for 1 h, about half of the sugar is in active cells and half in quiescent cells.

Glucose Transport Stimulation in Thymocytes by Human Albumin
As seen in Fig. 1, the presence of fatty acid-free albumin in the efflux medium caused a larger fraction of cellular methylglucose to exit in the fast phase, showing that many of the quiescent cells quickly exhibited transport activity comparable to that of active cells. It can be seen in Fig. 1 and other figures that about 10-20% of methylglucose exited slowly after exposure to fatty acid-free albumin, indicating that 60-80% of the quiescent cells were stimulated. Most of the remaining 20-40% were not stimulated with doses as high as 3% (data not shown). Fig. 1 also shows that an unextracted preparation of albumin did not stimulate. We have also failed to observe stimulation with fatty acid-free or unextracted bovine albumin from Sigma and Collaborative Research, Inc.
We were deceived by results of the kind shown in Fig. 1 into thinking that stimulation and nonstimulation are characteristics of extracted and unextracted human albumin, respectively. In fact, there is a great deal of lot-to-lot variability. We have not found another charcoal-extracted preparation as active as 70F-9310, and we have found only two other unextracted preparations (ICN 103704, lot 11276; Sigma A1653, lot 81F-9389) which may be as inactive as 30F-02271. Of six unextracted human albumins tested, one from Sigma (A2386, lot 121F-0277) was strongly stimulatory. Perhaps, strong stimulation will be found more frequently among charcoal-extracted preparations and lack of stimulation will be found more frequently among unextracted preparations, as our experience would suggest, but we must conclude that human albumin preparations typically stimulate glucose transport in thymocytes.
Adding back oleic acid and linoleic acid to charcoal-extracted human albumin in various fatty acid/albumin ratios did not reduce the stimulatory activity, so we could not attribute the stimulatory action to fatty acid poverty per se.
The fatty acid removal process (17) involved acidification with HC1 in the presence of charcoal, centrifugal removal of charcoal, and reneutralization with NaOH. We have not been able to elicit the stimulatory activity by acidifying (pH 3) and neutralizing human fraction V (Sigma A2386, lot 30F-02271), by alkalizing (pH 11) and neutralizing human fraction V, or by subjecting human fraction V to the entire fatty acid-re-moval procedure (using Darco charcoal or Norit-A and Millipore fitration to remove traces of charcoal remaining after centrifugation).
Dose a n d Time Dependences of Glucose Transport Stimulation by Fatty Acid-free Human Albumin- Fig. 2 shows the effects of various concentrations of albumin included in the medium with which the suspension was diluted to initiate exit. Submaximal doses stimulated a smaller number of cells.
In this experiment, 0.1% albumin resulted in an obvious stimulation, and 0.05% resulted in a questionable stimulation. Fig. 3 shows the results of several experiments in which 0.6% albumin was added after the fast exit phase was complete (active cells equilibrated by efflux). Efflux from quiescent cells was stimulated fully without any visible delay, judging from the linear extrapolation of the stimulated time course back to the control time course at the point of albumin addition.
Partial Destruction of Stimulatory Activity by Trypsin-Suspecting that contaminating insulin-like activity might be responsible for the stimulation, we treated the albumin with trypsin according to a procedure (18) reported to destroy insulin-like activity in albumin preparations. In fact, trypsin treatment reduced the stimulatory activity in a time-dependent and dose-dependent manner (Fig. 4).
Gel electrophoresis revealed that the main contaminant was a protein considerably larger than albumin. It was completely destroyed by trypsin treatments which affected only a minor fraction of the stimulatory activity, so we believe that this major contaminant is not the stimulant. It will be seen below that the agent is, in fact, a minor component of the albumin preparation.
Lack of Stimulation by Known Anabolic Hormones-Since glucose transport stimulation by serum fractions is usually attributed to insulin-like activity, we tested insulin and several anabolic factors for ability to stimulate methylglucose efflux. None of these agents stimulated when present in the medium with which the suspensions were diluted to initiate efflux. The dose ranges tested were bovine insulin, 1-100 nM; rat T lymphocyte growth factor, 0.2-2 units/ml; mouse epidermal growth factor, 1-600 ng/ml; multiplication-stimulating activity, 10-3000 ng/ml; and rat sulfation factor, 0.02-0.2 units/ml. Attempts to Separate Stimulatory Activity from Albumin-   We attempted to separate the stimulatory activity from albumin by gel filtration on a variety of bed types with a variety of media. These included Sephadex G-50 with incubation medium, Sephadex G-50 with 30 mM acetic acid, Sephadex G-50 with 900 rn acetic acid and 150 mM NaC1, Sephadex G-50 with 155 rn acetic acid and 155 mM NaCl, Sephadex G-100 with 155 mM acetic acid and 155 mM NaC1, Sephadex G-200 with 155 mM acetic acid and 155 mM NaC1, and Sephadex G-200 with incubation medium. In each case, the stimulatory activity activity eluted in the albumin peak. Stimulatory activity coincided with the albumin peak also on DEAE-Sephadex chromatography (A-50-120; 1.5 X 12 cm; gradient elution with 75 ml of 15 mM Tris-C1 (pH 7.2) and 75 ml of 15 m~ Tris, 15 mM acetic acid, 20 mM NaCl brought to pH 3 with HC1). These methods failed to distinguish the main stimulatory agent from albumin.
Effects of Thiol Reagent on Stimulatory Activity-Using the method of Grassetti and Murray (12), one can titrate about 1 thiol group/2 or 3 albumin molecules with 4-PDS. A 1% albumin solution, then, contains about 0.1 mM reactive thiol groups. Such thiol compounds as cysteine, glutathione, and dithiothreitol at concentrations less than 0.1 mM stimulate glucose transport dramatically and quickly by a mechanism which involves oxidation by molecular oxygen and is mediated by the resulting H202 (10). Although it seemed unlikely that such a reactivity would be exhibited only by the thiols in albumin (present at less than 1 group/molecule) and that such a reactivity, if present, could survive chromatography in oxygen-containing media, we decided to test the possibility by blocking the sulfhydryl groups of albumin. Blocking agents would, of course, react with sulfhydryl groups of any contaminating protein, so the broader question examined was whether reactive thiols are essential to the stimulatory agent.
Preliminary studies showed that fatty acid-free and untreated human albumin contained the same amount of 4-PDSreactive thiols, 0.3 group/molecule. However, the fatty acidfree albumin reacted somewhat faster, and the reaction went to completion with a slightly shorter half-time. Fatty acid-free albumin samples were then treated with excess thiol reagents (6PDS, benzoquinone, and N-ethylmaleimide) and then passed through Sephadex  columns equilibrated with incubated medium to remove the excess reagents. 4-PDS reduced stimulatory activity slightly both times it was tested. Benzoquinone reacted quickly with the albumin, producing a pink product characteristic of thio-substituted benzoquinone (19). This did not reduce stimulatory activity. Likewise, al- Mercaptoethanol was added to 1-ml portions in various molar ratios, and the mixtures were incubated 1 h at 37 "C and then passed through Sephadex G-50 columns (1.5 X 18 cm) equilibrated with incubation medium. The three 2-ml fractions from each column with greatest 280-nm absorbance were combined and diluted with medium to 0.25% albumin. Of this, 5 ml was used as the dilution medium to initiate efflux. The residue was examined from N-['4C]ethylmaleimide binding and 4-PDS reactivity. By these criteria, the lowest doses of mercaptoethanol (Mercupt.) (A, A which obliterated stimulatory activity) increased protein sulfhydryl groups slightly, whereas larger doses increased protein sulfhydryl groups in a proportional manner. MeGlc, methylglucose.

Glucose Transport Stimulation in Thymocytes by Human Albumin
kylation of most of the sulfhydryl groups with excess Nethylmaleimide failed to diminish stimulatory activity. The slight effect of 4-PDS may have been mediated by thiopyridine released in the reaction with protein thiols (see below). By contrast to thiol-reactive reagents, thiol compounds did obliterate stimulatory activity. Mercaptoethanol obliterated activity when added at low molar ratios (Fig. 5). The result suggested that the active agent contained an essential disulfide and was present in a molar ratio to albumin less than 0.1. In order to get a better upper limit estimate of the ratio of critical disulfides to albumin molecules, we treated the albumin prep-  aration with dithiothreitol in several molar ratios to albumin. As seen in Fig. 6, 1 dithiothreito1/100 albumin molecules abolished half of the stimulatory effect, and 1 dithiothreitol/ 50 of albumin abolished virtually all of the stimulatory effect. Some of the reagent would have been oxidized by molecular oxygen, and another portion would have reacted with the numerous disulfides of unstimulatory albumin molecules, so we suspect that the stimulatory agent is present at much less than 0.01 molecule/albumin molecule. Since stimulation was pronounced with 0.1-0.2% albumin, the agent is effective at concentrations less than 3 a W 7 M.

Tests for Possible Roles of Ca2+
and HzOz-When the cells were prepared in Ca"-free medium, loaded with [I4C]methylglucose in Ca2+-free medium containing EDTA equivalent to ?4 of the Mg2+ and 1 p g / d of A23187, and then diluted to initiate efflux in a similar medium, the stimulatory effect of fatty acid-free human albumin was as potent as usual (Fig. 7). These nearly lytic doses of A23187 in a Ca2+-free medium should have greatly upset the physiological control of Ca2+ compartmentation. We, therefore, think it unlikely that Ca2+ redistribution is a signal mediating the response.
In other experiments, fatty acid-free human albumin was compared with dithiothreitol as regards the effects of catalase on stimulation (Fig. 8). In these experiments, 20 p~ dithiothreitol stimulated as did 0.2% (30 PM) albumin. Catalase present in the diluting medium blocked stimulation by dithiothreitol but antagonized stimulation by albumin only slightly. In another experiment with a series of lower catalase concentrations, 16 pg/ml of catalase blocked stimulation by dithiothreitol and had no effect on Stimulation by albumin. If H202 is involved in albumin stimulation, it is not generated in the bulk medium.

DISCUSSION
The present studies show that many commercial human albumin preparations contain a factor(s) which stimulates glucose transport in rat thymocytes. Thus far, the most stimulatory preparation we have encountered was charcoal extracted for fatty acid removal, and the least active (inert) preparations were not so treated. However, a wide range of activities was found among treated and untreated prepara-

Glucose Transport Stimulation in
Thymocytes by Human Albumin 1194 1 tions, so we doubt the significance of charcoal extraction in this regard. We do not know whether the stimulatory agent(s) in various albumin preparations are the same or similar, and we do not know whether the one(s) in lot 70F-9310 is typical. That the agent might be altered albumin is suggested by our inability to separate it from albumin on gel filtration and ion exchange columns. That the agent might be a hormonelike peptide or protein rather than altered albumin is suggested by the fact that so few molecules bear the activity. It seems that a treatment (storage in whole blood, fractionation, extraction) to which all molecules are subjected might affect a larger fraction of them similarly. The upper limit estimate of 1 active molecule/50 albumin molecules may be a gross overestimate. Although the disulfides of the stimulatory agent are particularly susceptible to reduction by mercaptoethanol and dithiothreitol, a substantial fraction of reducing agent must have been consumed by disulfides in unstimulatory albumin molecules. That albumin disulfides react readily was seen by the "stoichiometric" production of protein thiols in the dithiothreitol treatment (with dithiothreitol doses which were significant relative to pre-existing protein thiols).
The thymocyte response appears to be a rather specific assay for certain unidentified stimulatory agents, for neither insulin, sulfation factor, nor any purified anabolic agent which we have so far been able to test would mimic the effect of stimulatory human albumin preparations.
Stimulation by albumin is unlike that of plant lectins and A23187 (11) and like that of H202 and N-ethylmaleimide (10) in that it does not appear to involve Ca2+ translocation. Catalase concentrations which block stimulation by dithiothreitol hardly affect stimulation by albumin, whereas much higher catalase concentrations appeared to interfere somewhat. This suggests a role of Hz02 generated at the plasma membrane, from which diffusion to its site of action would be favored over collision with extracellular catalase (by comparison with H202 generated from dithiothreitol in the bulk extracellular fluid).
From the beginning of our studies of glucose transport in thymocytes, we have used 0.2% bovine fraction V in our medium with the thought that this would reduce the likelihood of mechanical injury of the cells. We have been through numerous lots without seeing evidence of stimulation. After finding the stimulatory effect of fatty acid-free human albumin, we also tested charcoal-extracted (two lots) and unextracted bovine albumin at higher concentrations (0.4-3%) with negative results. We, therefore, believe stimulatory activity to be much more prevalent among human albumin preparations than bovine albumin preparations. Since glucose transport stimulation is a common action of growth factors, the stimulatory agent in human albumin might behave as a growth factor in some circumstances. However, the transport stimulant which we assay is not essential to the growth-promoting action of albumin in lymphocyte tissue culture (20-23). The latter is attributable to albumin per se, and it is very consistent among albumin preparations within a species and among species.