(Received for publication, August 30, 1995; and in revised form, March 4, 1996)
From the
To determine the role of Ras-dependent signaling pathways in
adipocyte function, we created transgenic mice that overexpress
Ha-ras in adipocytes using the aP2 fatty acid-binding
protein promoter/enhancer ligated to the human genomic ras sequence. ras mRNA was increased 8-17-fold and Ras
protein 4-5-fold in white and brown fat, with no overexpression
in other tissues. The subcellular distribution of overexpressed Ras
paralleled that of endogenous Ras. [U-C]Glucose
uptake into isolated adipocytes was increased
2-fold in the
absence of insulin, and the ED
for insulin was reduced
70%, with minimal effect on maximally stimulated glucose transport.
Expression of Glut4 protein was unaltered in transgenic adipocytes, but
photoaffinity labeling of transporters in intact cells with
[
H]2-N-[4-(1-azi-Z,Z,Z-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-propylamine
revealed 1.7-2.6-fold more cell-surface Glut4 in the absence of
insulin and at half-maximal insulin concentration (0.3 nM)
compared with nontransgenic adipocytes. With maximal insulin
concentration (80 nM), cell-surface Glut4 in nontransgenic and
transgenic adipocytes was similar. Glut1 expression and basal
cell-surface Glut1 were increased 2-2.9-fold in adipocytes of
transgenic mice. However, Glut1 was much less abundant than Glut4,
making its contribution to transport negligible. These in vitro changes were accompanied by in vivo alterations including
increased glucose tolerance, decreased plasma insulin levels, and
decreased adipose mass. We conclude that ras overexpression in
adipocytes leads to a partial translocation of Glut4 in the absence of
insulin and enhanced Glut4 translocation at physiological insulin
concentration, but no effect with maximally stimulating insulin
concentrations.
Insulin regulates a myriad of cellular events including
mitogenesis, gene expression, and various metabolic pathways such as
glucose transport, glycogen synthesis, and triacylglyceride synthesis.
Insulin stimulates glucose uptake into target tissues primarily via the
translocation of Glut4 from intracellular vesicles to the plasma
membrane(1, 2, 3) . Although major strides
have been made in elucidating signaling pathways involved in
insulin's regulation of gene expression and mitogenesis, the
molecular basis for insulin-stimulated glucose transporter
translocation remains elusive. Recent data indicate that activation of
PI ()3-kinase is involved, but PI 3-kinase activation alone
is not sufficient to achieve insulin-stimulated glucose
transport(4, 5, 6, 7, 8, 9, 10) .
The binding of insulin to its receptor initiates a cascade of signaling events involving activation of the receptor's intrinsic tyrosine kinase activity, receptor autophosphorylation, and phosphorylation of cellular substrates such as insulin receptor substrate-1 and -2 and Shc (11) . These proteins, in turn, act as docking modules for other proteins via SH2 domains, thereby forming multiple signaling complexes. One such signaling complex involves the interaction of insulin receptor substrate-1 and/or Shc with Grb2 and SOS, leading to the activation of the low molecular weight GTP-binding protein Ras. Insulin binding to its receptor causes an increase in cellular concentrations of GTP-bound Ras(12, 13, 14) , and signaling pathways emanating from Ras have been implicated in mediating at least a portion of insulin's pleiotropic effects on cell growth and metabolism(15) . However, the role of Ras signaling pathways in the regulation of insulin-stimulated glucose transport remains controversial.
Van den Berghe et al.(16) compared
the ability of insulin, epidermal growth factor, and thrombin to
stimulate the Ras/mitogen-activated protein kinase cascade in 3T3-L1
adipocytes and found that Ras activation alone is not sufficient for
insulin-stimulated glucose transport. Osterop et al.(12) and Draznin et al.(17) also reported
that enhanced Ras signaling in Rat-1 cells had no effect on
insulin-stimulated glucose transport. In contrast, Kozma et al.(18) reported that transfection of 3T3-L1 adipocytes with
constitutively activated Ras resulted in enhanced translocation of
Glut4 to the plasma membrane in the absence of insulin, and insulin did
not further stimulate glucose uptake or Glut4 translocation.
Interpretation of the latter study is tempered by the fact that Ras
overexpression resulted in an 95% down-regulation of Glut4
expression. Recently, transient expression of constitutively active Ras
in primary rat adipocytes led to increased levels of cotransfected
epitope-tagged Glut4 at the cell surface(10) . Further support
for this phenomenon comes from studies in which microinjection of
activated Ras into cardiac myocytes caused a significant increase in
glucose transport(19) . In contrast, microinjection of
constitutively active Ras (20) or downstream effectors such as
activated Raf (21) into 3T3-L1 adipocytes had no effect on the
translocation of Glut4. Additionally, injection of neutralizing
antibodies directed against Ras resulted in a 50% inhibition of
insulin-stimulated glucose transport in cardiac myocytes (19) ,
but had no effect in 3T3-L1 adipocytes(20) . The latter
observation is supported by the fact that transfection of rat
adipocytes with dominant-negative Ras did not affect translocation of
epitope-tagged Glut4(10) . Recently Reusch et al.(22) found that lovastatin, which inhibits farnesylation
of Ras (and thus appropriate targeting to the plasma membrane),
attenuated insulin effects on thymidine incorporation and glucose
incorporation into glycogen, but not glucose transport. However, the
effects of lovastatin may not be specific to Ras since it also inhibits
the rate-limiting step of cholesterol biosynthesis(23) ,
geranylgeranylation of proteins (23) , insulin stimulation of
PI 3-kinase activity(24) , and cell transformation by
Raf(25) .
We have addressed the question of Ras involvement in insulin-stimulated glucose transport using an in vivo approach, by developing transgenic mice that overexpress Ha-Ras exclusively in brown and white adipose tissue. We show that wild-type Ras overexpression leads to a partial translocation of Glut4 and Glut1 in the absence of insulin as well as increased sensitivity to the effects of insulin on Glut4 translocation. However, Ras overexpression does not alter maximally insulin-stimulated glucose transport, suggesting that at maximal insulin concentration, Ras is not rate-limiting for glucose transport in adipocytes.
Figure 1: Schematic representation of the transgene construct. 5.4 kilobases of mouse genomic DNA containing the mouse aP2 fatty acid-binding protein promoter/enhancer were fused to 6.4 kilobases of the human Ha-ras genomic DNA as described under ``Experimental Procedures.''
Figure 2: Adipose-specific overexpression of human Ha-ras in two lines of transgenic mice. RNA was extracted, and Northern blotting was performed as described under ``Experimental Procedures.'' A, Northern blot comparing relative abundance of Ha-ras mRNA in brown adipose tissue from transgenic mice overexpressing ras at a high or moderate (Mod) level. 20 µg of total RNA were run on each lane. B, Northern blot showing tissue distribution of transgene mRNA in transgenic mice overexpressing ras at moderate levels. 20 µg of total RNA were run on each lane. T designates mice heterozygous for the transgene, and N designates nontransgenic mice. Bat, brown adipose tissue; Wat, white adipose tissue.
Fig. 3A (upper panel) shows that the overexpression of Ras at the protein level in white adipose tissue was also severalfold higher in the high level overexpressing line compared with the moderate line. The exposure shown was chosen to illustrate the difference in the levels of Ras protein in the two transgenic lines and thus is too light to see Ras protein in nontransgenic fat. However, Ras protein could be observed in nontransgenic lanes with longer exposure. Fig. 3A (lower panel) shows a representative blot of Ras protein in post-nuclear membranes prepared from isolated adipocytes from nontransgenic and moderate level Ras-overexpressing mice. The mean of multiple blots shows that Ras protein is increased 3-4-fold in the moderate level overexpressing mice compared with nontransgenic mice. No adjustments need to be made to compare the amount of Ras expression per cell since the recovery of total membrane protein was not different in isolated adipocytes from nontransgenic and transgenic mice (nontransgenic, 447 ± 99 pg/cell; transgenic, 595 ± 66 pg/cell; n = 4, p = not significant). Levels of Ras protein could not be assessed in isolated adipocytes from high level overexpressing mice due to the difficulty in harvesting large amounts of isolated adipocytes from this line. However, the quantitative information in the moderate overexpressing line is most important since that is the line in which we pursued both in vivo and in vitro studies of insulin action.
Figure 3:
A,
relative abundance of p21protein in perigonadal
white adipose tissue and isolated adipocytes from moderate (Mod) and high (Hi) level Ras-overexpressing
transgenic mice. B, subcellular localization of Ras in
adipocytes from nontransgenic and moderate level Ras-overexpressing
mice. Western blotting was performed and quantified as described under
``Experimental Procedures.'' A, post-nuclear
membranes were prepared from adipose tissue from nontransgenic mice and
from moderate level and high level Ras-overexpressing transgenic mice (upper panel) and from isolated adipocytes from nontransgenic
and moderate level Ras-overexpressing transgenic mice (lower
panel). Adipose tissue data (upper panel) are from
individual animals and reflect determinations on three to four
mice/group. Data for isolated adipocytes (lower panel)
represent two separate determinations, each performed on adipocytes
pooled from 15 to 30 mice/group. B, subcellular fractions of
isolated perigonadal adipocytes (homogenate, high density microsomes (HDM), low density microsomes (LDM), and plasma
membranes (PM)) from nontransgenic (Non Tg) or
moderate level Ras-overexpressing transgenic (Tg) male mice
(12-24 weeks of age, 15-30 mice/group) were prepared as
described under ``Experimental Procedures.'' Prior to
fractionation, cells were incubated in the absence(-) or presence
(+) of 80 nM insulin for 30 min. Due to low protein
recovery, high density microsome fractions from both +insulin and
-insulin treatments were combined
(-/+).
This similarity in the relative distribution of Ras
between nontransgenic and transgenic cells is one indication that the
membranes from the nontransgenic and transgenic cells are fractionating
in a similar manner. As an independent assessment of this, we measured
cytochrome c reductase activity, a marker for the endoplasmic
reticulum. High density microsomes from both nontransgenic and
transgenic cells were enriched 3-fold in cytochrome c reductase activity, whereas low density microsomes and plasma
membranes showed no enrichment in both genotypes. Recoveries of
cytochrome c reductase activity were similar for both
genotypes. Adequate membrane protein was not available for additional
marker enzyme assays.
Gonadal fat pad weight was reduced with both moderate and high level Ras overexpression in both male and female mice compared with nontransgenic mice. Because expression of the transgene had marked effects on gonadal fat pad weight and cell size at all ages and the effects were of similar magnitude from 10 to 24 weeks in males and from 12 to 20 weeks in females, the data for the combined ages are shown in Table 1. In male mice, there was a ``dose-dependent'' effect of the transgene, so an even greater reduction in fat pad weight was seen with high level Ras overexpression compared with moderate overexpression. Adipocyte size was reduced in both male and female transgenic mice compared with nontransgenic mice (p < 0.05). High level overexpression tended to have a greater effect on adipocyte size than moderate level overexpression (p < 0.05 in females, but not significant in males). Grossly, fat pad morphology was normal with moderate level Ras overexpression, but strikingly different with high level Ras overexpression where increased vascularity was observed. This, coupled with the very small adipocyte size, made floatation and isolation of adipocytes from the high level overexpressing line very difficult. Thus, only limited studies could be carried out using the high level overexpressing line.
Figure 4: Glucose tolerance tests in transgenic mice with moderate level Ras overexpression and nontransgenic litter mates. After an overnight fast, D-glucose (1 mg/g of body weight) was injected intraperitoneally in awake mice, and blood glucose was determined on samples from the tail vein. Data are means ± S.E. for four male mice in each group. Mice were 10 weeks old. Areas under the curves are different at p < 0.01.
Figure 5:
Insulin dose-response curves for glucose
transport into isolated adipocytes from nontransgenic and moderate
level Ras-overexpressing mice. Adipocytes were isolated and incubated
with varying concentrations of insulin as described under
``Experimental Procedures.'' Left panel, glucose
transport expressed as amol/cell/min. Right panel, data after
curve fitting, which corrects for differences in basal rates of
transport. Data are means ± S.E. of quadruplicate samples for a
single experiment using male nontransgenic (open squares) and
moderate level Ras-overexpressing transgenic (closed squares)
mice that were 14 weeks of age. Curves are representative of seven
experiments for nontransgenic and six experiments for transgenic male
mice that were 12-14 weeks of age (see Table 3). Right
panel inset, ED values calculated by curve fitting.
Values for ED
are means ± S.E. for seven
nontransgenic (NTG) and six transgenic (TG) male mice
that were 12-14 weeks of age. *, different from nontransgenic
mice at p < 0.04.
Figure 6:
Insulin dose-response curves for glucose
transport into isolated adipocytes from nontransgenic and high level
Ras-overexpressing mice. Adipocytes were isolated and incubated with
varying concentrations of insulin as described under
``Experimental Procedures.'' Left panel, glucose
transport expressed as amol/cell/min. Right panel, data after
curve fitting, which corrects for differences in basal rates of
transport. Right panel inset, ED values
calculated by curve -fitting. Values for both panels and the inset are means ± S.E. for three nontransgenic (NTG; open squares) and three transgenic (TG; closed
squares) female mice that were 12 weeks of age. *, different from
nontransgenic mice at p < 0.05.
The
difference in the ED values for the controls in Fig. 5and Fig. 6is due to the fact that different sexes
were studied. Normal female mice are more sensitive to insulin than
males, both in vivo and in adipocytes in
vitro(27) . By studying both sexes, we demonstrate that
the increased insulin sensitivity is present in adipocytes from
Ras-overexpressing mice of both sexes ( Fig. 5and Fig. 6and Table 3).
Figure 7: A, relative abundance of Glut4 and Glut1 proteins in perigonadal white adipose cells from nontransgenic (N) and moderate level Ras-overexpressing transgenic (T) mice. Post-nuclear membranes were prepared, and Western blotting was performed and quantified as described under ``Experimental Procedures.'' Data shown are for pooled cells from 15 to 30 male mice/group. Mice were 12-24 weeks of age. This blot is representative of three separate Western blots. B, photolabeling of cell-surface Glut4 (closed squares) and Glut1 (open squares) in isolated adipocytes from moderate level Ras-overexpressing transgenic mice. Adipocytes were isolated, incubated without (Basal) or with (Insulin Stimulated) a maximally stimulating concentration of insulin, and photolabeled with ATB-BMPA as described under ``Experimental Procedures.'' Data are expressed as dpm/gel slice and are representative of two separate experiments, each performed on cells from five to nine male mice/group. Mice were 12-16 weeks of age.
Fig. 8shows a representative experiment in which photolabeling of plasma membrane Glut4 in the basal state was 2-fold greater in adipocytes from moderate level Ras-overexpressing mice compared with nontransgenic mice. This enhanced translocation of Glut4 in the absence of insulin was mirrored by a 2.8-fold increase in basal glucose transport in transgenic adipocytes. When data from three experiments were meaned (cells pooled from a total of n = 20 nontransgenic mice and n = 26 transgenic mice), we observed a 2.4 ± 0.4-fold increase in photolabeling and a 2.2 ± 0.3-fold increase in glucose transport in transgenic adipocytes compared with nontransgenic adipocytes (p < 0.05). Similar results were observed when Glut4 was measured by immunoblotting plasma membranes prepared from isolated adipocytes (data not shown).
Figure 8: Comparison of glucose transport and photolabeling of cell-surface Glut4 in intact adipocytes from nontransgenic and moderate level Ras-overexpressing transgenic mice. Adipocytes were prepared, and glucose transport (hatched bars) and photolabeled Glut4 (solid bars) were measured in the absence of insulin and with half-maximal insulin concentration (0.3 nM) as described under ``Experimental Procedures.'' Data are from adipocytes from six to eight male mice/group (12 weeks of age). All data are expressed per cell.
To determine if the enhanced sensitivity of glucose transport to insulin (Fig. 5) was due to enhanced translocation of Glut4, photoaffinity labeling with the bismannose photolabel was conducted after adipocytes were incubated with half-maximal insulin concentration (0.3 nM) (Fig. 8). Half-maximally insulin-stimulated glucose transport and photolabeled Glut4 were approximately 2-fold higher than in nontransgenic cells.
The cellular mechanism(s) that regulate insulin-stimulated
Glut4 translocation remain largely unknown. Insulin stimulates the
activation of p21, and the Ras/mitogen-activated protein
kinase pathway has been shown to regulate many of the effects of
insulin on mitogenesis and protein synthesis(15) . However, the
role of Ras in insulin-stimulated glucose transport remains
controversial. Data are conflicting as to whether pathways emanating
from Ras are important or even involved in insulin stimulation of Glut4
translocation. Using a transgenic approach, we examined the role of
p21
in the regulation of Glut4 translocation in adipose
tissue. Mice that overexpress wild-type Ha-Ras exclusively in adipose
tissue have higher rates of basal glucose transport and increased
insulin-sensitive glucose transport in adipocytes. The mechanism
appears to be insulin ``mimicking,'' with enhanced
translocation of Glut4 to the plasma membrane in the absence of insulin
and in response to submaximal, but not maximal, concentrations of
insulin.
The fact that wild-type Ras (which is not structurally modified to be constitutively active) has this
insulin-like effect is somewhat unexpected since Ras activation is
generally thought to require ligand binding to a receptor. The effects
we observe suggest that Ras can be activated to some degree in the
absence of ligand binding. Support for this possibility comes from the
observation that p21 colocalizes with
cell-surface receptors (42) and may facilitate
oligomerization, leading to receptor activation independent of ligand
by cross-phosphorylation(43) . Furthermore, our results
indicate that the amount of Ras protein is rate-limiting for activation
of signaling for Glut4 translocation in the absence of insulin and at
submaximal insulin concentrations, but not at maximally stimulating
insulin concentrations.
Our results are entirely consistent with reports of the effects of activated Ras either transfected into rat adipocytes (10) or microinjected into cardiac myocytes(19) . In the former study, cell-surface Glut4 was increased in the absence of insulin, and in the latter, basal glucose transport increased with no change in Glut4 gene expression. Finally, chronic overexpression of constitutively active Ras in 3T3-L1 adipocytes (18) also increased cell-surface Glut4 in the absence of insulin; however, interpretation of these data is compromised by the 95% reduction in Glut4 expression in these cells.
Others have shown that the Ras/mitogen-activated protein kinase
pathway is not sufficient or necessary for insulin-stimulated glucose
transport or Glut4 translocation by overexpressing activated
Ras(20) , dominant-negative Ras(20) , or activated Raf (21) in 3T3-L1 adipocytes or by treating these cells with a
specific mitogen-activated protein kinase kinase
inhibitor(44) . We also have data, in another transgenic mouse
model using Asn dominant-negative Ras, that signaling via
Ras is not essential for maximally insulin-stimulated glucose
transport(45) . In contrast, PI 3-kinase-mediated signaling has
recently been shown to be necessary for insulin-stimulated glucose
transport, albeit not
sufficient(4, 5, 6, 7, 8, 9, 10) .
The requirement for PI 3-kinase signaling in the regulation of Glut4
translocation does not necessarily preclude a role for Ras since the
Ras and PI 3-kinase pathways have recently been shown to potentially
converge(46, 47, 48) . Taken together with
the data in the literature, our current study indicates that more than
one pathway may be capable of eliciting Glut4 translocation in the
absence of insulin and at submaximal insulin concentrations. Additional
evidence for more than one pathway comes from the fact that Glut4
translocation stimulated by exercise and hypoxia does not appear to
involve PI 3-kinase as it is not sensitive to the PI 3-kinase
inhibitor, wortmannin(49) .
Transgenic mice overexpressing
wild-type Ras have reduced adipocyte size, and one might question
whether this could affect glucose transport rates. The changes in
glucose transport in the adipocytes from the Ras-overexpressing mice
are unlikely to be accounted for by the smaller cell size since studies
in rodents have shown that smaller adipocytes have lower basal
glucose transport rates and no change or lower transport in response to
submaximal and maximal insulin stimulation(50, 51) .
In this study, we observe higher basal and submaximally
insulin-stimulated transport rates in the smaller cells from transgenic
mice. In addition, our own data on nontransgenic FVB mice, the strain
used for this study, show no correlation between cell size and either
the ED for insulin-stimulated glucose transport or the
maximal insulin-stimulated glucose transport rate (data not shown).
Thus, the increase in basal and submaximally insulin-stimulated glucose
transport in adipocytes from Ras transgenic mice cannot be explained by
differences in cell size.
The changes in glucose transport also
cannot be attributed to effects on Glut1 expression in adipocytes.
Insulin is known to regulate the expression of
Glut1(20, 52) , and this appears to be via the
p21/mitogen-activated protein kinase
pathway(20) . In our study, moderate level overexpression of
wild-type Ras in fat of transgenic mice results in a 2-fold
overexpression of Glut1 in adipose tissue. However, this increase in Glut1 gene expression does not explain the enhanced glucose
transport in adipocytes from transgenic mice since Glut1 is much less
abundant than Glut4(37, 53) , and our photolabeling
data show that this is true even in Ras-overexpressing adipocytes.
Furthermore, Glut1 has a 3-fold lower turnover number (mol of glucose
transported per transporter/time) than Glut4(54) . Thus, Glut1
appears to play a very minor role in glucose transport into the
adipocyte in both nontransgenic and transgenic mice.
The effects of Ras overexpression at submaximal insulin concentrations are physiologically relevant since these concentrations fall in the normal range for circulating insulin levels. Thus, the effects of Ras overexpression on glucose transport in adipocytes may explain, at least in part, the apparent increased glucose tolerance (Fig. 4) and enhanced sensitivity to insulin (Table 2) in the Ras overexpressing transgenic mice in vivo. Although it may be surprising that increased glucose uptake selectively into adipose tissue can affect whole body insulin action, this is supported by our recent studies showing enhanced glucose tolerance and increased insulin sensitivity in transgenic mice overexpressing Glut4 only in adipose tissue(27) . The direct uptake of glucose into fat may account for only a part of the enhanced glucose disposal in Ras-overexpressing mice. However, the fact that glucose transport into skeletal muscles from these mice is not altered in vitro makes it unlikely that chronic alterations in muscle account for the enhanced insulin sensitivity in vivo. Although leanness is often associated with increased insulin sensitivity and obesity is associated with insulin resistance, these changes usually persist in skeletal muscle in vitro(55) . In our model, enhanced insulin sensitivity may be present in muscle in vivo due to changes in the hormonal/metabolic milieu. Thus, future studies will determine whether the effects on insulin action in vivo in the Ras overexpressing transgenic mice could also be due to secondary changes in circulating levels of substrates (i.e. free fatty acids) that are known to affect glucose uptake or peptides (tumor necrosis factor and leptin) whose synthesis or release may be affected by altering Ras expression.
These studies also clearly establish a role for Ras in adipocyte development and maintenance in vivo. Previous evidence indicated that Ras might be important for adipocyte differentiation. Transfection of a dominant-negative mutant form of Ras into 3T3-L1 preadipocytes resulted in inhibition of differentiation to adipocytes (13) . Thus, one might expect that overexpression of Ras in adipocytes of transgenic mice would foster adipocyte differentiation, and the enhancement of glucose transport would increase lipogenesis, resulting in increased adipose mass. However, we observe reduced gonadal fat pad weight and reduced adipocyte size in transgenic mice with moderate levels of Ras overexpression, and this effect is enhanced with high levels of Ras overexpression. The possibility that Ras plays an important role in the regulation of adipose mass is strengthened by our preliminary observation of increased adipose mass in transgenic mice overexpressing dominant-negative Ras in adipocytes(45) . Future investigations will determine whether this results from direct effects of Ras in adipocytes or indirect effects due to changes in the levels of circulating substrates and hormones that affect metabolism and eating behavior. For example, lower circulating insulin levels (Table 2) may result in increased lipolysis and decreased lipogenesis in vivo. The possibility that these changes in adipose mass could be mediated by leptin, the protein product of the newly cloned ob gene(56) , is currently under investigation.
In conclusion, while signaling emanating from Ras is not essential for maximally insulin-stimulated glucose transport, overexpression of wild-type Ras in adipocytes of transgenic mice results in increased basal and submaximally insulin-stimulated glucose transport. The mechanism involves increased cell-surface Glut4. The in vivo consequences of increased wild-type Ras expression include reduced adipose tissue mass and increased insulin sensitivity.