Membrane vesicles from brown adipose tissue: A ... - Bioscience Reports

Bioscience Reports, VoL 12, No. 2, 1992

Membrane Vesicles from Brown Adipose Tissue" A Tool for the Study of Amino Acid Transport. The Case of L-Alanine A . R o d r i g u e z - M a r t i n , ~ N . B e P a n d X . N e m e s a r L2 Received February 26, 1992

A density gradient method is used to isolate membrane vesicles from brown adipose tissue. These respond to changes in osmolarity and show the classical overshoot pattern when L-alanine uptake is assayed. Transport is shown to be effected by two components: a tinear (Ka =0.498 min 1) and Na+-dependent saturable component (Km = 2.3 raM) and a Vm~x = 19.9 pmol/~tg protein • rain). This pattern is similar to that shown by cells isolated from brown adipose tissue. KEY WORDS: alanine; brown adipose membrane vesicles; transport. ABBREVIATIONS: MeAIB; Methyl-aminoisobutyric acid.

INTRODUCTION A general picture of amino acid transport systems has e m e r g e d from the study of different cell lines in various animal species, which differentiates between widely distributed systems and those found in only one cell type (1, 2). Experiments usually involve the use of metabolite inhibitors or structural analogues (3, 4) in order to block the normal metabolism of substrates inside the cell, since the kinetic properties of transport systems may be modified by the accumulation of substrate. The use of isolated m e m b r a n e vesicles from tissues avoids these metabolic interferences and m a y eludicidate the kinetic properties of transport systems (5). This procedure has been applied to the study amino acid transport in liver (6), intestinal brush border (7), kidney and other tissues (8, 9). Although few studies of amino acid transport kinetics in brown adipose tissue have been reported (10), our recent results (11) suggest that L-alanine transport may be mediated by similar systems to those described in other cell types. In order to determine whether or not the kinetic p a r a m e t e r s measured in isolated Unitat de Bioquimica i Biologia Molecular ]3. Departament de Bioqu~mica i Fisiologia, Universitat de Barcelona. 08071 Barcelona. Spain. 2To whom correspondence should be addressed, 115 0144-8463/92/04~-0115506.50/0 ~) 1992 Plenum Publishing Corporation

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cells are influenced by the inhibition of metabolism of L-Alanine, we have attempted to isolate membrane vesicles from brown adipose tissue, with a procedure normally used to obtain liver vesicles.

MATERIAL

AND

METHODS

Adult female Wistar rats (210-230 g) were housed under standard temperature (22 + 1°C), light (12 hours on/12 hours off) and humidity (70%). Animals were decapitated and after exsanguination, the interscapular brown adipose tissue was gently removed (avoiding all the white adipose tissue), weighed, placed in cold buffer (0.25 mM sucrose, 0.2 mM CaCI2 of 10 m M H E P E S , pH = 7.5) and carefully minced with scissors. The same procedure was followed with the liver, that served as a control. The following procedure was based on that described by Van Amelsvoort et al. (5) and modified by ourselves (12). Briefly, the tissue was gently homogenized, in a Potter-Elvehjem homogenizer in cold buffer, and then filtered through nylon cloth. The filtrate was diluted fourfold with buffer supplemented with E D T A (in a final concentration of I raM) and centrifuged at 30,000 x g for 20 min. The pellet was resuspended in buffer with EDTA and centrifuged at 700 x g for 10 min. Supernatant was collected and mixed with isotonic Percoll (Pharmacia), at 11% final proportion, and centrifuged at 30,000 x g for 30 min. The membrane vesicles band was collected and washed with EDTA-free buffer, with 2 cycles of resuspension-centrifugation at 30,000 x g for 30 min. Finally, the pellet was resuspended in buffer, quickly frozen in liquid nitrogen and stored at -40°C. Vesicles stored up to for one month maintained their total capacity for transport assay. Protein in homogenate and membrane preparations was determined according to (13). 5'-Nucleotidase (E.C.3.1.3.5) activity was selected as a plasma membrane marker and assayed in both fractions (14). The enzyme markers of membranes of other subcellular organelles were assayed in both homogenate and in plasma membrane vesicle preparations: cytochrome-oxidase (E.C. 1.9.3.1) as a mitochondrial marker (15); 13-N-acetyl-glucosaminidase (E.C. 3.2.1.30) as a lysosomal marker (16) and glucose-6-phosphatase (E.C. 3.1.3.9) as a marker of endoplasmic reticulum (17). The activity of NADPH-cytochrome c reductase (E.C. 1.6.2.4) was also assayed as marker of endoplasmic reticulum (18). The transport procedure was adapted from that of Sips et al. (6) as previously described (12). Briefly, 10/21 of membrane vesicles was mixed with 40/tl of the incubation mixture, giving the following concentrations: 0.25mM sucrose, 0.2 mM CaC12, 10 mM H E P E S / K O H (pH = 7.4), 100 mM either Na +- and K +sulphocyanate and L-alanine from 0.1 to 20 mM, supplemented with L-(2, 3)3H alanine. The specific activity of alanine was monitored to obtain a medium with 20-50 KBq/~mol in all cases. The intravesicular medium was the same as the incubation mixture but without alanine, Na +- and K+-sulfocyanate. The reaction was stopped after 5 seconds (near linearity condition) by putting 20/tl of the mixture into a tub containing l ml of cold stop solution (0.25M Sucrose, 100 mMNaC1, 0.2 mM CaC12, 10 mM H E P E S / K O H = 7.4). Then, the entire

Alanine transport by membrane vesicles

117

contents was quickly filtered through a nitrocellulose filter (Schleicher & Schtill, Germany) (pore size 0.45/~m) and washed with 4 ml of the cold stop solution. Dried filters were counted for radioactivity. All the assays were carried out in triplicate. The incubations were performed at 25°C. The amounts of alanine taken up by plasma membrane vesicles were calculated from the radioactivity retained by the filters and the specific activity of the medium. The inhibitory effect of M e A I B on L-Alanine uptake was achieved by the addition of M e A I B to the incubation medium, at a concentration twenty times higher than that of L-alanine. The inhibitory action of other amino acids on L-alanine uptake was also studied by adding these amino acids to the incubation medium at 20 mM concentration. 2,3-3H-L-Alanine was purchased from the Radiochemical Centre (Amersham, U.K.). Other reagents (high quality grade) were obtained from Sigma (St. Louis, USA) and Merck (Darmstad, Germany). Kinetic analysis were performed using the program F I G P (Fig P Coporation, USA). Statistical comparisons were carried out with the Student's t test.

RESULTS Table 1 shows the specific activities of the marker enzymes in the isolated vesicles. The activity of the enzymes in brown adipose tissue are in the same range as those of the liver (data not shown). The recovery of 5'Nucleotidase activity in the vesiculate fractions in relation to homogenate activity was in the same order as the recovery of N A D P H - r e d u c t a s e and gtucose-6-phosphatase, the values of the other enzyme activities being lower. Furthermore, the enrichment of marker enzyme activities followed the same pattern, the plasma membrane fraction being the most enriched. Figure 1 shows the alanine transport by m e m b r a n e vesicles as a function of time. Thus, the response was more marked in a Na+-rich than in a K+-rich medium. Maximal activity was obtained at 5 seconds. On the other hand, this overshoot shows less activity (three times less) than that of liver vesicles obtained by the same procedure (data not shown). The membrane vesicles of brown adipose Table 1. Specificactivities of the membrane marker enzymes in isolated membrane vesicles of brown adipose tissue

5'Nucleotidase Glucose-6-phosphatase g-N-acetyl-glueosaminidase Cytochrome oxidase NADPH-reductase

Specific activity

Recovery

Enrichment

6.9 + 3.7 1.2 5:0.08 0.7 ± 0.1 304 5:10 45 5:3.1

3.0 + 0.7 2.6 ± 0.3 0.5 ± 0.1 0.07 5:0.01 3.9 5:0.6

2.6 + 0.6 1.6 5:0.5 0.9 :t: 0.1 0.09 5:0,01 1.4 5:0.4

Activities are expressed as gmol/h/mg of protein. Recoveries are expressed as percentage of the original activity in the homogenate recovered in the membrane preparation. Enrichment values are calculated as the ratio of the specificactivity in plasma membrane preparations and in the homogenates.

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1 / osmolarity Fig. 1. a) L-alanine uptake of L-alanine by membrane vesicles from brown adipose tissue in response to time. 0 : Na÷-rich medium, A: K÷-rich medium. The results are the mean d: s.e.m, of three experiments, b) Response of the concentrative capacity of membrane vesicles to hyperosmolarity. The results are the mean + s.e.m, of three different experiments. The incubations were performed at 25°C for 30 minutes.


119

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Alanine Fig. 2. Concentration-dependent L-alanine uptake by m e m b r a n e vesicles from brown adipose tissue. 0: total transport in N a + - m e d i u m , &: transport in a K§ m e d i u m , []: Total transport-transport in a K+-rich m e d i u m . T h e results are the m e a n + s . e . m , of three different experiments. The incubations were performed at 25~

tissue showed a linear response to changes in the reciprocal of medium osmolarity. Thus, the uptake of L-alanine (expressed as pmol//~g of protein) in a variable osmolarity (ranging from 0.25 to 2 osmols) showed a good linearity, with a correlation coefficient = 0.8912). The mean apparent vesicular volume of brown adipose isolated membrane vesicles was 216 :t: 45 nl/mg protein. Figure 2 shows the kinetics of alanine uptake in membrane vesicles of brown adipose tissue. The total transport can be decomposed in two components; a linear component, in a K+-rich medium that can be assumed as a diffusion component (Kd = 0.498 min -~) and a saturable component, obtained by substraction of the linear component from the total transport. Table 2 shows the values of the kinetic parameters of the saturable Table 2,

Vm. x K M (raM)

Kinetic parameters of the saturated L-alanine Na+-dependent transport in m e m b r a n e vesicles from brown adipose tissue TOTAL

MeAIB-sensitive

MeAIB-insensitive

19.9 + 1.3 2.3 :t: 0.7

17.8 + 0.9 2 . 9 + 0.41

5.7 + 1.7 3 6 + 1.2

Vma x activities are expressed

as pmol Ala/pg of protein, min. The values are the

m e a n + s.e.m, of three different experiments.

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Table 3. Inhibition of Na +dependent L-alanine uptake in mem-

brane vesicles from brown adipose tissue Alanine Methyl AIB Cysteine Leucine Glutamine

100 + 1.1 46 + 2.1" 47 + 1.5" 4.9 4-0.3* 27 4- 1.1"

The activity of L-alanine transport are expressed as % of activity detected when transport was performed with 1 mM alanine and 20 mM amino acid concentration. Basal value= 6.9 pmolAla//~g protein/min.*= P-< 0.05 vs. basal values component of alanine transport in membrane vesicles of brown adipose cells. The saturable component of total transport shows a Vmax of 19.9pmol Ala//zg of protein-min and a KM = 2.3 mM. From a parallel experiment performed in a similar medium as described and supplemented with 20 mM MeAIB, the kinetic pattern of MeAIB-sensitive and MeAIB-insensitive saturable transport can be deduced. Thus, the Vmax values were of the same magnitude as those of total transport, and the MeAIB-insensitive transport showed a higher KM value than total and MeAIB-sensitive transport. Table 3 shows the percentage of alanine transport detected in the presence of 20 mM concentrations of several amino adds. Thus, MeAIB and Cys inhibited the transport by around 50%, while the inhibition created by Gln, and specially by Leu was, much higher.

DISCUSSION

The application of a method designed for the isolation of vesicles from liver basolateral membranes, has been shown in brown adipose tissue, in the light of the validation of results obtained in isolated cells, in order to obtain a methodological device with which to study the elevated potential capacity to uptake amino acids shown by this tissue (10), Thus, there is a clear response in the uptake of L-alanine into vesicles in relation to time which is higher in the Na+-rich than in K+-rich medium. This pattern is as expected, since the uptake of L-alanine in most cell types, and in other membrane vesicles, is mediated by the A and ASC systems, which are Na+-dependent. However, the overshoot shows a lower uptake capacity than liver vesicles (12), although the response is fast, since the maximal uptake ratio is accomplished at 5 seconds. This reduced uptake capacity could be explained by the presumably significant proportion of endoplasmic reticulum membrane in the vesicle composition, since the enrichment in the activity of the plasmatic membrane marker (5'-Nucleotidase) represents, at least, 2.5 times the activities


121

of the endoplasmic markers (glucose-6-phosphatase and NADPH-reductase), in contrast with the higher ratio described in liver vesicles (5-6 times) (12). The enrichment values obtained are lower than those described in the processes designed for the isolation of purified membrane functions, but not for that of the vesicle obtention (19, 20). However, there is a relative purification of membrane fractions, since the lysosomal and especially the mitochondrial fractions, show low enrichment. Furthermore, the results confirm that the utilization of glucose-6phosphatase as a marker of the endoplasmic reticulum is useful, since the enrichment is the same as that obtained from NADPH-reductase. The functionality of membrane vesicles is confirmed both by the concentrative response in relation to time (overshoot) and by the response of the vesicles to the medium osmolarity, since the response is linear. Thus we can assess that isolated membrane vesicles from brown adipose tissue can be useful for the determination of the transport pattern of several metabolites. The pattern of L-alanine transport follows that described in isolated brown adipose cells. Thus, the total transport can be divided into a Na+-dependent saturable component and a linear component, the linear component being the predominant pattern, which could be explained by the heterogenous composition of membrane vesicles. However, the analyss of kinetic parameters, reveals a Km value, in the same order of magnitude, although higher than those showed by isolated cells (11), yet similar to those described in isolated vesicles of liver (21, 22). Analysis of saturable component of transport in the presence of MeAIB, an inhibitor of system A transport (23), shows different responses, both in capacity and in affinity, in the MeAIB-sensitive and -insensitive components that act in the L-alanine transport. Thus, on the basis of the action of the transport systems described in cells or isolated vesicles (6, 23), we could assign the activity of MeAIB-sensitive component to system A, and the MeAIB-insensitive component, to system ASC. Thus, the presence of both transport systems, with an equivalent potentiality in the saturable L-alanine transport (as confirmed by the 50% inhibition in the transport generated both by MeAIB and Cys), is present at the end of purification process, in spite of the possible contamination with other membrane types, confirming the pattern shown in the isolated cells (11). However, the specificity of components of the L~atanine transport described, seems to be low, since the high inhibition in L-alanine transport caused by amino acids as leucine (that is transported mainly by the L system) or glutamine, allow to speculate about the broad capacity of these systems, as well as the importance of the diffusion component, which represents 50% or more of the total transport. Thus, the procedure used is valid to obtain membrane vesicles from brown adipose cells, although the main inconvenience may result from the low amount of this tissue that can be obtained from a rat (about i g per rat).

ACKNOWLEDGEMENTS This work was supported by a grant from Direcci6n General de Investigacidn Cientffica y T6cnica (PB88-0208) from Spanish Government. Thanks are given to Robin Rycroft for his help in the correction of the manuscript.

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Rodriguez-Martin, Bel and Remesar REFERENCES

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