THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 258! No. 12, Issue of June 25. pp. 7425-1429,1983
Printed In U.S.A.
Proposed Mechanism of Insulin-resistant Glucose Transport in the
Isolated Guinea Pig Adipocyte
SMALL INTRACELLULAR POOL OF GLUCOSE TRANSPORTERS*
Richard Horuk$#, Martin Rodbell#, Samuel W. Cushmann, and LawrenceJ. Wardzalan
From the §Laboratory of Cellular and Deuelopmental Biology, and Wellulur Metabolism and Obesity Section, National Znstitute
of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205
A marked resistance to the stimulatory action of
insulin on glucosemetabolism has previously been
shown in guinea pig, compared to rat, adipose tissue
and isolated adipocytes. The mechanism of insulin re-
sistance in isolated guinea pig adipocytes has, there-
fore, been examined by measuring 12’I-insulin binding,
the stimulatoryeffect of insulin on 3-0-methylglucose
transport and on lipogenesis from [3-’H]glucose, the
inhibitory effect of insulin on glucagon-stimulated
glycerol release, and the translocation of glucose trans-
porters in response to insulin. The translocation of
glucose transporters was assessed by measuring the
distribution of specific D-glucose-inhibitableI’HJcyto-
chalasin B binding sites among the plasma, and high
and low density microsomal membrane fractions pre-
pared by differential centrifugation from basal and
insulin-stimulated cells.
At a glucose concentration (0.5 mM) where transport
is thought to be rate-limiting for metabolism, insulin
stimulates lipogenesis from 30 to 80 fmol/cell/90 min
in guinea pig cells and from25 to 380 fmol/cell/90 min
in rat cells with half-maximal effects at approximately
100 p~ in both cell types. Insulin similarly stimulates
3-0-methylglucose transport from 0.40 to 0.70 fmol/
cell/min and from0.24 to 3.60 fmol/cell/min in guinea
pig andrat fatcells, respectively.
Nevertheless, guinea pig cells bind more insulin per
cell than rat cells, and insulin fully inhibits glucagon-
stimulated glycerol release. In addition, the differences
between guinea pig and rat cells in the stimulatory
effect of insulin on lipogenesis and 3-0-methylglucose
transport cannot be explained by the greater cell size
of the formercompared to the latter(0.18 and 0.09 fig
of lipid/cell, respectively). However, the number of
glucose transporters in the low density microsomal
membrane fraction prepared from basal guinea pig
cells is markedly reduced compared to that from rat
fat cells (12 and 70 pmol/mg of membrane protein,
respectively) and the translocation of intracellular glu-
cose transporters to the plasma membrane fraction in
response to insulin is correspondingly reduced. These
results suggest that guinea pig adipocytes are mark-
edly resistant to the stimulatory action of insulin on
glucose transport and that this resistance is the cone-
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “aduertisernent” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
4 TO whom all correspondence should be addressed a t Bldg. 6,
Room B1-13, NIH, NIADDK, Bethesda, MD 20205.
7425
This is an Open Access article under the CC BY license.
(Receivedfor publication, February 8,1983)
quence of a relative depletion in the number of intra-
cellular glucose transporters.
In contrast to normal rat adipose tissue and isolated fat
cells, guinea pig adipose tissue and isolated adipocytes have
previously been shown to be markedly resistant to the stim-
ulatory effect of insuIin on glucose metabolism (1, 2). The
resistance to insulin observed at glucose concentrations in the
range of those considered to be normal fasting plasma levels,
approximately 5 mM, is probably the consequence ofthe low
capacity for de novo fatty acid synthesis from glucose in guinea
pig cells (1, 2). However, a marked insulin resistance is still
observed at those low glucose concentrations (less than 0.5
mM) where transport is thought to represent the rate-limiting
step for metabolism (2). Thus, guinea pig adipose cells may
also be resistant to the stimulatory effect of insulin at the
glucose transport level, a fundamental step in the regulation
of glucose metabolismby insulin (3-5).
Recent studies with isolated adipocytes (6-12) and dia-
phragm (13, 14) from rats indicate that insulin stimulates
glucose transport by inducing a rapid and reversible change
in the distribution of glucose transporterstothe plasma
membrane from what appear to be intracellular membranes
having different properties from the plasma membrane.
Studies of this insulin-induced “cycling” of glucose trans-
porters have been limited so far to tissues or cells from the
rat, in part because of the large magnitude of insulin action
on glucose transport in this experimental animal. However,
in several insulin-resistant states, thestreptozotocin-induced
diabetic rat (15), the high fat-fed rat (16), and theaged, obese
male rat (17), decreasedglucose transport inresponse to
insulin correlates with a decreasedpool of intracellular glucose
transporters, suggesting that glucose transport in these ani-
mals is limited by the quantity of glucose transporters avail-
able fortranslocation to theplasma membrane. Thus, a major
defect in animals displaying insulin resistance, at least with
respect to glucose utilization, appears to be changes in the
concentration of glucose transporters. In order to determine
whether similar defects in glucose transport could account for
the insulin resistance of guinea pig adipocytes,we examined
the stimulatory effects of insulin on glucosetransport activity
and on the distribution of glucose transporters in guinea pig
adipocytes and compared these effects with those observed in
isolated rat adipocytes studied in parallel. In addition, we
have examined several other parameters of insulin action
such as insulin binding, the Stimulatory effect of insulin on
lipogenesis from glucose at a low glucose concentration and
the inhibitory effect of insulin on glucagon-stimulatedlipol-
7426 Insulin-resistant Glucose Transport in Guinea
Adipocytes
Pig
ysis. Based on these studies we conclude that the poor re- ent from that observed in the rat adipocyte. This suggests
sponsiveness of guinea pig adipocytes to insulin can & ex- thatthe insulin receptor is functional in the guineapig
plained by a reduction in the number of intracellular glucose adipocyte. Indeed, insulin is as potent in inhibiting the lipo-
transporters. lytic effect of glucagon on the guinea pig adipocyte as has
been reported for the antilipolytic action of insulin in the rat
MATERIALS AND METHODS adipocyte. As shown in Table I, the half-maximal inhibition
Male Sprague-Dawley rats (140-170 g) and NIH strainguinea pigs of glucagon-stimulated lipolysis occurs between 0.1 to 1 nM
(150-700 g) were obtained from Murphy Breeding Labs, Plainfield, insulin, which isconsistent with the concentration range over
IN. Animals were fed laboratory chow ad libitum and were sacrificed which insulin inhibits lipolysis in rat adipocytes (32).
by decapitation.
Cytochalasins B and E were from Sigma, ‘“I-labeled insulin and The presence of functional receptors is confirmed in Fig. 2
all other radiochemicals were from New England Nuclear, crude which illustrates that the specific binding of ‘261-insulinto
collagenase (type 1) was from Worthington, and bovine serum albu-
la
min, fraction V, was from Reheis Chemical Co. Tris and all other .-
P
reagent grade chemicals were from Sigma. .-a
Isolation of Adipocytes and Determination of Adipocyte Size and
Numbr-Isolated adipocytes were obtained by enzymatic digestion
of epididymal fat pads from rats and guinea pigs as described by
Rodbell (18) and modified by Cushman and Wardzala (7). Cell size
and number were determined by the osmic acid fixation method
originally described by Hirsch and Gallian (19) for intact tissue
fragments and modified for isolated fat cells by Cushman and Salans
(20).
Glucose Transport-Glucose transport was assayed in isolated adi-
pocytes by measuring the rapid uptake of 3-O-[methyl-3H]glucoseas
described by Gliemann and Whitesell (21) and modified by Foley et
al. (22). For experimental conditions, see Table 11. Insulin concentration (MI
Insulin Binding-Isolated adipocytes (2 X 10‘ cells/ml) suspended
in Krebs-Ringer-phosphate buffer, pH 7.4, containing 1%alhumin, FIG. 1. The conversionof [3-sH]glucose(0.55 mM)into lipid
were incubated with bovine ‘=I-insulin (100PM) and native insulin by isolated rat )(. and guinea pig (0)adipocytes stimulated
(0 to 1PM) in plastic minivials in a shaking water bath at 24 ‘C for with increasing concentrations of insulin. Data from at least
90 min. Cells and medium were separated by oil centrifugation (23). three experiments +. S.E. See “Materials and Methods” for experi-
Nonsaturable binding was determined in thepresence of excess native mental conditions.
insulin (1 &M).
Lipolysis-Adipocyte lipolysis was determined by enzymatic assay TABLEI
of glycerol release (24) from glucagon-stimulated fat cells. For exper- Inhibition of glucagon-stimulatedglycerol releasein isolated guinea
imental conditions, see Table I. pig fat cells by increasing concentrations of bovine insulin
Lipogenesis-Lipogenesis was determined by following the incor-
poration of ~-[3-~H]glucose into lipid as described by Moody et al. Fat cells (2 X IO5 cells/ml) were incubated with shaking at 37 “C
(25). Briefly, adipocytes (2 X 10’ cells/ml) were incubated with for 90 min in 400 p1 of Krebs-Ringer bicarbonate medium, pH 7.4,
shaking at 37 “Cfor 90 min in 1 ml of medium containing D-glucose containing 1%defatted albumin, glucagon (100 nM), and varying
(0.55 mM), ~-[3-~H]glucose (0.1 &i), and bovine insulin (0to 1nM).
concentrations of insulin as indicated. After incubation 600 g1 of
At the end of the incubation, the adipocytes were extracted into a water were added and the cells were homogenized in a small glass
toluene-based scintillant a t room temperature for 1 h,andthen hand-held homogenizer (10 up and down strokes) and transferred to
counted. Eppendorf tubes for centrifugation at 12,000 X g for 8 min. The clear
Preparation of Subcellular Fractions and Determination of the infranatant was withdrawn and heated to 70 “C for 5 min. Aliquots
Number of Glucose Transporters-Subcellular fractions from isolated were removed and glycerol was determined (24).
adipocytes were prepared by differential ultracentrifugation as out- Inhibition of glucagon-
lined by Karnieli et al. (8).Fluoride-sensitive adenylate cyclase activ- Insulin concentration Glycerol stimulated lipolysis by
insulin
ity was determined using the method of Salomon et al.(26). 5’-
Nucleotidase activity was determined using the method of Avruch nhf nnwl/lOs cells/h %
and Hoelzl-Wallach (27), rotenone-insensitive NADH-cytochrome c 20 20.0 100
reductase activity was determined using the method of Dallner et ai. 4 25.1 88
(28), and UDP-ga1actose:N-acetylglucosamine galactosyltransferase 1.o 37.6 60
activity was determined using the method of Fleisher (29). Protein 0.1 50.0 25
was determined by the method of Lowry et al. (30), as modified by 0.01 53.0 la
Peterson (31). The number of D-glucose-inhibitable cytochalasin B 0 64.0 0
binding sites inthe subcellular fractions was determined as previously
described (7).

RESULTS
The comparative effects of bovine insulin in stimulating
Iipogenesis from [3-3H]glucose are shown in Fig. 1 for isolated
adipocytes fromguinea pigs and rats. At the low extracellular
glucose concentrations used in this study (0.5 mM),transport
is thought to represent the rate-limiting step in glucose me-
tabolism (4, 22). In the absence of insulin, the conversion of
glucose to lipids in guinea pig adipocytes is slightly higher
than that observed in rat adipocytes. In contrast, the maximal
conversion of glucose to lipids induced by insulin in the guinea FIG. 2. The displacementof Ia5I-labeledinsulin from guinea
pig adipocyte is 80 fmol/cell/90 min which is less than 20% pig (0)and rat Q adipocyte insulin receptors in the Presence
of that seen in rat adipocytes, 380 fmol/cell/90 min. Note, of increasing concentrationsof unlabeled insulin.Data from at
however, that the range of insulin concentrations required for least three experiments f S.E. See “Materials and Methods” for
stimulating lipogenesis in guinea pigadipocytes is not differ- experimental conditions.
 Insulin-resistant Glucose Transport in Guinea Pig Adipocytes 7427

guinea pigadipocytes is morethan %fold greater than thatto

rat adipocytes although half-maximal binding is observed at
approximately the same insulin concentrations (4 to 6 nM).
Even when the larger cell size of the guinea pig adipocyte
(Table 11) is taken into account, specific binding per unit of
cellular surface area is still
greater in theguinea pig, than rat,
adipocyte. Other characteristics of guinea pig and rat insulin
receptors were examined; for example, the pH optimum for
insulin binding was 7.8 for both species of adipocytes and, as
reported previously forthe rat adipocyte, insulin binding was
abolished when guinea pigadipocytes were treated with tryp-
sin under the same conditions (data not shown). Multiplica-
tion-stimulating factor, a growth factor that binds poorly to
the insulin receptor in rat adipocytes (33), also displayedthe
same relative displacement of lBI-insulin from the guinea pig
adipocyte receptor (data not shown). From these findings,
therefore, the guinea piginsulin receptor shares similar prop-
erties with those of the rat insulin receptor and is at least
comparable in quantity.
The comparative effects of insulin on 3-0-methylglucose
uptake, a measure of glucose transport activity (2l), in FIG. 3. The distributionof marker enzymeactivities in sub-
guinea pig and rat adipocytes are shown in Table 11. In the cellularfractionsisolated from basal and insulin-treated
basal state, 3-0-methylglucosetransport is somewhat greater guinea pig fat cells. Plasma membrane (PM), high density micro-
in the former than in the latter. However, in contrast to the somes (HDM), low densitymicrosomes (LDM). A, 5'-nucleotidase
activity; B, sodium fluoride-stimulated adenylate cyclase activity;C,
15-foldeffect of insulin seen with rat adipocytes, insulin NADH-cytochrome c reductase activity;D, UDP-ga1actose:N-acetyl-
caused no more than a %fold effect on 3-0-methylglucose glucosaminegalactosyltransferase activity. The uerticalbar repre-
transport in the guinea pig adipocyte. Thus, glucose transport sents S.E.; data are from two separate experiments. The open boxes
parallels lipogenesis in the presence of 0.55 mM glucose in the represent membranes from basal cells, and the hatched boxes repre-
guinea pig adipocyte, suggesting that glucose transport may sent membranes from insulin-treatedcells.
be a limiting factor in the amount of glucose utilized by the
guinea pig adipocyte, even though the insulin receptors are effect on 3-0-methylglucosetransport.
sufficient in quantity and apparently fully functional. Since In the rat adipocyte, glucose transport correlates closely
age and nutritional status can affect responsiveness of adi- with the number of glucose transporters present in the plasma
pocytes to insulin (34), we also examined the effects of insulin membrane; insulin appears to act primarily by stimulating
on guineapig adipocytes isolated from animals that were the translocation of glucosetransporters from an intracellular
younger and lower in body weight than the animals routinely membrane compartment to the plasma membrane(6-12).
used inthese studies (600-700 9). As shown inTable 111, even Therefore, we investigated whether the glucose transporters
in younger animals insulin did not exert more than a %fold in guinea pig adipocytes are distributed between the plasma
membrane and intracellular membranes in a manner that
TABLEI1 could account for the different basal and insulin-stimulated
3-0-methylglucose uptake by rat and guinea pig adipocytes rates of sugar transport relative to that observed with rat
Isolated adipocytes were incubated in the absence or presence of adipocytes.
insulin (7 nM)for30 min at 37"C. Uptake was initiated by the The fractionation scheme for separating various membrane
addition of 50 p1 of a mixture of ~-[l-'~C]glucose and 3 - O - [ ~ t h y l -
'HJglucose (final concentrations,58.2and 11.6 pCi/pmol,respec-
fractions was the same as that employed for rat adipocytes
tively) to 400 r l of cell suspension. Uptake was terminated by the (8):fractions designated plasma membrane, and high and low
addition of 20 pl of a 10 mM solution of cytochalasin B. Cells and density microsomal membrane fractions were prepared and
medium were separated by oil centrifugation and the net uptake of analyzed for their content of the marker enzymes used to
3-0-methylglucose was calculated as described previously (8). Data characterize these fractions in rat adipocytes (Fig. 3). As in
from three separate experimentsf S.E. the case of rat adipocytes, the plasma membrane fraction in
3-0-Methylglucose transport the guinea pig adipocyte is enriched in both 5'-nucleotidase
AdipOCyteS Cell size
Basal Insulin and fluoride-sensitive adenylate cyclase activities; the high
pg lipid/ceU fmollceU/rnin density microsomal fraction is also enriched in the endo-
Rat 0.09 0.24 k 0.05 3.6 f 0.18 plasmic reticulum marker, rotenone-insensitive NADH-cyto-
Guinea pig 0.18 0.40 k 0.09 0.7 f 0.10 chrome c reductase (7).The low density microsomal fraction,
characterized in the ratadipocyte as being enriched in UDP-
TABLE111 ga1actose:N-acetylglucosamine galactosyltransferase activity
3-O-~thy&lucoseuptake by guinea p C adipocytes prepared from
(lo), a Golgi membrane marker, displays a lowerspecific
150- to 250-g animals activity than does the plasma membrane fraction. This dif-
Data fromtwo experiments f S.E. See legend to Table I1 for ferencein UDP-ga1actose:N-acetylglucosaminegalactosyl-
experimentalconditions. transferase distribution precludes a direct comparisonbe-
tween the guinea pig and rat adipocyte membrane fractions
%
:$ Cell size
3-0-Methylglucose transport
Basal Insulin
with respect to their relative content of membrane species.
As has been reported for rat adipocytes (71, insulin does not
g pg tipid/cell fmol/cei&nin alter the distribution of the various marker enzymes (Fig.3),
150 0.09 0.09 & 0.07 0.30 f 0.05 nor does insulin alter therecovery of protein in these fractions
200 0.09 0.48 f 0.08 1.10 f 0.17 (Table IV).
250 0.12 0.34 f 0.02 0.64 f 0.01
Specific D-glucose-inhibitable cytochalasin B binding was
7428 Insulin-resistant Glucose Tramport in Guinea Pig Adipocytes
TABLEIV
Protein recovery from subcellular fractions of guinea pig fat cells
Data from three experiments -+ S.E.
Protein recovery
Fraction
Basal Insulin
pglcell
Homogenate 880.0 k 130.0 880.0 k 90.0
Plasma membrane
High density microsomes
36.9 2 6.7
37.6 f 8.5
*
32.6 9.2
43.4 f 10.3
Low density microsomes 57.6 2 9.9 71.2 -C 14.8
GPig Rat G.Pig Rat G.Pig Rat
FIG. 4. The distribution of specific D-glucose-inhibitable
cytochalasin B binding sites in subcellular fractions from
basal and insulin-stimulated guinea pig fat cells. A, plasma
membranes; B , high density microsomes; C, low density microsomes.
The vertical bar represents S.E.; data are from three separate exper-
iments. The open boxes represent membranes from basal cells, and
the hatched boxes represent membranes from insulin-treated cells.
used to assess the concentration of glucose transporters in
these various membrane fractions (7). Fig. 4 shows the distri-
bution of glucose transporters in the three membrane frac-
tions of the guinea pig adipocyte and the effects of insulin
thereon. For direct. comparison, membrane fractions were
isolated in identical fashion from rat adipocytes and compared
for their glucose transporter concentration and the effects of
insulin. In thecase of the ratadipocyte the number of glucose
transporters/mg of membrane protein in the low density
microsomal membrane fraction in the basal state is approxi-
mately 9-fold greater than that in the plasma membrane
fraction; insulin reduces the number of transporters in this
fraction and stimulates a comparable enhancement in the
plasma membrane fraction, in confirmation of earlier studies
(7). In contrast, guineapig adipocytes in the basal state
contain fewer glucose transporters/mg of membrane protein
in thelow density microsomal membrane fraction than in the
plasma membrane fraction and insulin does not alter this
distribution. The plasma membrane and high density micro-
somal membrane fractions contain comparable numbers of
glucose transporters in both cell types in the basal state; the
numbers of glucose transporters in the latter fraction arenot
significantly influenced by insulin.
DISCUSSION
The results of this study indicate that insulin-resistant
glucose utilization in guinea pig adipocytes cannot be attrib-
uted to a lack of insulin receptors or to an inability of these
receptors to function when occupiedby hormone. The guinea
pig adipocyte binds approximately 3 times more insulin than
the ratadipocyte with similar apparent dissociation constants
7 and 5nM, respectively (Fig.2). Moreover, to theextent that
insulin stimulates glucose utilization and acts as anantilipo-
lytic agent, its potency in guinea pigs is comparable to that
found for rat adipocytes. Guinea pig insulin, which differs
structurally from the bovine insulin used in this study, is no
more effective than the Iatter in stimulating glucose utiliza-
tion in guinea pig adipocytes (35). The characteristics of the
guinea pig insulin receptor investigated here further suggest
that theinsulin receptors in guinea pigand ratadipocytes are
not fundamentally different in either structureor function.
It has been previously demonstrated that the capacity of
guinea pig adipocytes to metabolize glucose at the low sugar
concentrations used in thisstudy (0.1 mM) is not rate-limiting
(2). For this reason we consider it unlikely that the poor
stimulation of glucose metabolism by insulin in guinea pig
adipocytes reflects the low capacity of the cells to metabolize
glucose.
The relatively low responsiveness of guinea pig adipocytes
to insulin with respect to glucose metabolism seemsto reside
in processes beyond the receptor. The major difference de-
tected between the guinea pig and rat adipocytes that might
account for the large differences in glucose utilization ob-
served here between the two celltypes is the concentration of
glucose transporters.
As judged from the rates of 3-0-methylglucose transport
under maximally stimulating concentrations of insulin, the
rat adipocyte may have upto 5 times more glucosetransport-
ers than the guinea pig adipocyte. However, the dissociation
constant for the D-glucose-inhibitable cytochalasin B binding
sites are almost identical in the plasma membrane fractions
derived fromthe adipocytes of the two cell types, 100 and 105
nM, respectively.
Based on cytochalasin B binding to the three major mem-
brane fractions isoIated from guinea pig and rat adipocytes,
the differences in glucose transporter number are on the order
of 4-fold, whichis consistent with the differences in maximal
levels of lipogenesis from glucose (cf. Fig. 1). However, this
binding assay includes the total number of glucose transport-
ers in the three fractions and does not account for those
transporters that are strictly available for glucose transport
in the intact adipocyte, i.e. those in the plasma membrane.
Unfortunately, we have no way of knowing how many of the
glucose transporters in the plasma membrane fraction are
actually associated with this membrane. The finding that
UDP - galactose :N-acetylglucosamine galactosyltransferase
activity, which is not usually a marker for the plasma mem-
brane, is also enriched in this fraction indicates caution in
interpretation. However, it should be noted that UDP-galac-
tose:N-acetylglucosamine galactosyltransferase is not a spe-
cific marker of glucose transport activity in the low density
microsomal membrane fraction (11).In addition, if the high
specific activity of UDP-ga1actose:N-acetylglucosamine ga-
lactosyltransferase in the plasma membrane fraction reflects
substantial cross-contamination of this fraction with low den-
sity microsomes, then there should also be a corresponding
increase in the number of glucose transporters/mg of mem-
brane protein in this fraction (derived from the intracellular
pool). The fact that the plasma membrane fractions of both
guinea pigand rat adipocytes contain comparable numbers of
glucose transporters (summed basal and insulin-stimulated
states) militates against such a view. This would also be
inconsistent with the very much lower rates of glucose trans-
port observed in the guinea pig adipocyte. Thus, in addition
to lower concentrations of transporters, the possibility should
be considered that thetransporters in the guinea pig adipocyte
are in different membrane structures from those in the rat
adipocyte that carry the transporters.
Our conclusion from the present study is that the guinea
pig, a normal animal showing typical glucose tolerance and
responses to insulin (36), contains adipocytes which are mark-
edly resistantto insulin at the level of glucose transport
compared to rat adipocyte. This insulin resistance appears to
be the consequence of having a lower concentration of glucose
transporters, similar to the streptozotocin-diabetic, high fat-
fed, and aged obese male rat (15-17). It remains to be seen
 Insulin-resistant Glucose Transport in Guinea Pig Adipocytes
whether this phenomenon involves decreasing the synthesis
of the transporters and/or altering their distribution between
the plasma membrane and intracellular membranes.
Acknowledgments-We wish to thank Mary Jane Zarnowski and
Dena R. Yver for their expert technical assistance during this work,
Dr. C. Londos for helpful discussions, and Bonnie Richards for the
typing of the manuscript.
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