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Research Service of the Department of Veterans Affairs, Denver Veterans Affairs Medical Center, Department of Medicine, University of Colorado Health Sciences Center (B.D., I.G., R.S., K.W., M.G.), Denver, Colorado 80220; San Diego Veterans Affairs Medical Center, and Department of Medicine, University of California (P.M., Y.K., J.O.), San Diego, California 92161; Department of Nutrition, Case Western Reserve University School of Medicine (J.F.), Cleveland, Ohio 44106; Diabetes Branch (M.R.) and Developmental Endocrinology Branch (D.A.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Veterans Affairs Medical Center and Department of Medicine, University of Mississippi Medical Center (R.C., D.M.), Jackson, Mississippi 39216
Address all correspondence and requests for reprints to: Dr. Boris Draznin, Veterans Affairs Medical Center (151), 1055 Clermont Street, Denver, Colorado 80220. E-mail: drazninb{at}den-res.org
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Abstract |
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To further investigate the role of acquired insulin resistance in modulating insulin’s effect on p21Ras prenylation, we induced insulin resistance in rats by glucosamine infusion. Insulin-resistant glucosamine-treated animals displayed significantly increased farnesylated p21Ras in response to insulin infusion compared to that in control saline-treated animals. Transgenic models of insulin resistance (heterozygous insulin receptor substrate-1 knockout mice, A-ZIP/F-1 fatless mice, and animals overexpressing glutamine:fructose-6-phosphate amidotransferase) contained increased amounts of farnesylated p21Ras.
We conclude that hyperinsulinemia, either endogenous (a prominent feature of insulin resistance) or produced by infusions of insulin, increases the amounts of farnesylated p21Ras in humans, mice, and dogs. This aspect of insulin action may represent one facet of the molecular mechanism of the potentially detrimental influence of hyperinsulinemia.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular mechanism of insulin signaling can be arbitrarily divided into two branches: metabolic and mitogenic. Even though there is a certain amount of cross-talk between the two branches, the former appears to involve phosphorylation of the insulin receptor substrate (IRS) family of proteins and activation of phosphatidylinositol 3-kinase and protein kinase B (5, 6). In contrast, the mitogenic branch of insulin action appears to engage the activation of the Ras, Raf, MEK, and mitogen-activated protein kinases (7) and propagate the insulin signaling toward the cell nucleus. Activation of the Ras pathway also leads to the phosphorylation and activation of the prenyl transferases: farnesyltransferase (FTase) and geranylgeranyltransferases I and II (GGTase I and II) (8, 9, 10). FTase and GGTase I and II posttranslationally modify a variety of cellular proteins, including a large family of small molecular weight GTPases (reviewed in Ref. 11). Post-translational modification of the GTPases is accomplished by the isoprenylation of conserved cysteine residues found on their C-termini (11, 12). Ras and Rho proteins are prenylated with farnesyl or geranylgeranyl on a single cysteine residue of the C-terminal CaaX box (where C = cysteine, a = aliphatic residue, and X = methionine, serine, or glutamine for Ras or leucine for Rho proteins) by FTase and GGTase I, respectively. In contrast, Rab proteins, which terminate with C-terminal cysteine motifs of CC or CxC, are double prenylated with geranylgeranyl by GGTase II (11, 12, 13, 14). As a group, prenyl transferases bind isoprenyl groups to their substrates, so that these GTPases may be anchored to their respective membranes, loaded with GTP, and thereby activated.
We have previously shown that the addition of insulin augmented FTase activity and the amount of farnesylated p21Ras in 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and vascular smooth muscle cells (9, 15, 16). Moreover, our data suggested that hyperinsulinemia, via augmentation of the amounts of farnesylated p21Ras, significantly amplified cellular nuclear responses to other growth factors, such as insulin-like growth factor (IGF-I), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) (8, 9, 15, 17). This priming effect of insulin required the presence of the intact insulin receptor (9) and was blocked by an inhibitor of FTase (8, 9, 15, 17).
Furthermore, in experiments with hyperinsulinemic animals, such as ob/ob mice and fa/fa Zucker rats, we found increased amounts of farnesylated p21Ras in liver, aorta, and skeletal muscle (17). In contrast, the levels of farnesylated p21Ras were normal in denervated rat skeletal muscle (17), a model of insulin resistance in the absence of hyperinsulinemia (18). Even though these findings suggested an association of hyperinsulinemia with increased amounts of farnesylated p21Ras, they did not establish a cause and effect relationship between these two variables. The present study was undertaken to confirm the causal role of hyperinsulinemia in both humans and experimental animal models.
Using normoinsulinemic controls and models of acquired or genetic insulin resistance, we demonstrate that hyperinsulinemia, resulting from either infusions of insulin or endogenous metabolic insulin resistance, increases the prenylation of p21Ras, thus creating a favorable background for enhanced mitogenic responsiveness of these tissues to various growth factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insulin infusion in mice
Male C 57BL/6 mice, aged 10–14 weeks, were fitted with chronic
indwelling jugular catheters according to the method of Ren et
al. (19) as previously described (20). Insulin (20 mU/kg·min)
was infused for 230 min while a steady state blood glucose level was
maintained. Animals were then killed by an overdose of anesthetic, and
the liver and aorta were removed, frozen immediately in liquid
nitrogen, and stored at -70 C. An equal number of animals was killed
before insulin infusion, and they served as controls.
Insulin infusion in dogs
Studies were conducted in 14 male mongrel dogs, weighing
20.8–28 kg. Animals were housed and operated upon as described
previously (21). Eight animals were made hyperinsulinemic by diverting
the veins of the pancreas from the portal vein to the vena cava (PVD
dogs) (21). A sham operation was performed on the control group of dogs
(n = 6). Hyperinsulinemic euglycemic clamp studies were performed
1 month postoperatively. Infusion of insulin (7.2 nmol/kg·min) was
continued for 160 min while maintaining steady state levels of
glycemia. Liver samples were obtained by biopsy before and at the
conclusion of the insulin infusion. Tissue was frozen in liquid
nitrogen and stored at -70 C.
Hyperinsulinemic euglycemic clamps in normal and obese
humans
Initially, white blood cells (WBC; buffy coat) were obtained
from 6 normal weight [4 men and 2 women; body mass index (BMI),
23.8 ± 0.66] and 21 obese (17 men and 4 women; BMI, 36.17
± 1.25) individuals (aged 30–50 yr) from an overnight fasting sample.
Subsequently, 4 normal weight and 4 obese individuals underwent
infusions of insulin (80 mU/m2·min) for 180
min. None of these individuals received any medications, none was
hypertensive, and none had a family history of diabetes. WBC were
obtained before and at the end of the insulin infusion. The cells were
frozen immediately and stored at -70 C until measurements of
farnesylated p21Ras were performed.
Glucosamine infused in rats
Animals were infused with either saline (0.5 ml/h) or
glucosamine (6.5 mg/kg·min) for 6.5 h as described previously
(22). Their preglucosamine or presaline insulin level was 10.0 ±
3.0 µU/ml and was not increased by the infusion of glucosamine
(11.5 ± 1.5 µU/ml). At 5 h, a hyperinsulinemic (4
mU/kg·min; to yield a plasma concentration of 
100 µU/ml)
euglycemic glucose clamp was performed for 90 min. The animals
were anesthetized, and tissue and blood samples were collected for
determinations of prenylated p21Ras, glycemia, and insulinemia
(22).
Transgenic models of insulin resistance
We have used three transgenic models of insulin resistance
developed, characterized, and previously described by the authors of
the current study. The heterozygous insulin receptor (IR)/IRS-1
knockout mice (50% reduction) have been developed in the laboratory of
Dr. Accilli (23) by breeding heterozygous IR knockout
(IR+/-) mice with heterozygous IRS-1 knockout
(IRS-1+/-) mice. The latter were obtained from
Dr. C. R. Kahn’s laboratory (24). The A-ZIP/F-1 mice were
developed in the laboratories of Drs. Reitman and Vinson (25), and the
glutamine:fructose-6-phosphate amidotransferase (GFA)-overexpressing
mice were developed in the laboratory of Dr. McClain (26). All three
models have been shown to be resistant to the metabolic actions of
insulin (23, 24, 25, 26, 27). Liver samples were used in the experiments with IRS-1
knockout and A-ZIP/F-1 mice. The GFA-overexpressing mice were infused
with insulin for 60 min, and the hind limb muscle samples were
collected for measurements of prenylated p21Ras as described below.
A-ZIP/F-1 mice were studied at birth and 3 weeks of age,
GFA-overexpressing mice were 6–10 weeks old, and
IRS-1+/- and IR/IRS-1+/-
mice were 6 months of age at the time of study.
Determinations of farnesylated p21Ras and geranylgeranylated
Rho-A
Tissue samples were homogenized in lysis buffer (150
mM NaCl, 5 mM MgCl2, 1
mM phenylmethylsulfonylfluoride, 1 mM
dithiothreitol, 1 mM sodium vanadate, 1 mM
sodium phosphate, 1% Triton X-100, 0.5% SDS, 10 µg/ml aprotinin, 10
µg/ml leupeptin, and 50 mM HEPES, pH 7.5). Crude lysates
were sonicated and centrifuged at 10,000 rpm. Total protein from the
resultant supernatant was determined by the bicinchoninic acid assay
(Pierce Chemical Co., Rockford, IL) and diluted to 1
mg/ml. Equal volumes of lysate and 4% Triton X-114 were combined in a
borosilicate glass tube, vortexed, and incubated at 37 C for 3 min.
Solutions were kept at room temperature until phases had separated.
Equal volumes from each phase were placed into separate 1.5-ml
microcentrifuge tubes, and p21Ras or Rho-A was immunoprecipitated using
a monoclonal anti-Ras antibody (Y13–259) or a monoclonal anti-Rho-A
antibody, respectively. Prenylated p21Ras and Rho-A were recovered in
the detergent phase (8, 9). Increases in the amounts of prenylated
proteins have been previously shown to reflect and closely correlate
with increases in the activity of prenyl transferases (8, 9, 10, 16).
Statistical analysis
All statistics were analyzed using Student’s t test,
with P < 0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B) and displayed
significantly (P < 0.05) increased amounts of
farnesylated p21Ras in their WBC (Fig. 1C; 53.8 ± 2.4%
vs. 45.2 ± 1.7%).
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).
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). Similarly, an infusion of insulin
raised the amounts of farnesylated p21Ras in the livers of normal dogs
(Fig. 4).
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).
Amounts of farnesylated p21Ras in experimental models of insulin
resistance induced by glucosamine
Our current (Figs 1 and 4) and previously published (16) data
indicate that the tissues of insulin-resistant animals and individuals
contain increased amounts of prenylated p21Ras. We postulated that
insulin resistance to the metabolic aspects of insulin action augments
the influence of the ensuing hyperinsulinemia on prenylation of the Ras
family of proteins. To confirm this hypothesis experimentally, we
examined the amount of prenylated p21Ras in tissues of animals with
acquired and genetic insulin resistance.
To induce insulin resistance experimentally, we infused male Sprague
Dawley rats (150–175 g) with either saline (0.5 ml/h) or glucosamine
(6.5 mg/kg·min) for 6.5 h followed by infusion of insulin (4
mU/kg·min) for 180 min (euglycemic clamp) as previously described
(22). Glucosamine infusion resulted in diminution of glucose disposal
as measured by euglycemic clamp (22). However, 6.5 h of
glucosamine infusion did not produce endogenous hyperinsulinemia
(11.5 ± 1.5 vs. 10 ± 3 µU/ml in saline-infused
rats) and did not affect the amount of farnesylated p21Ras (48 ±
3.2% vs. 46 ± 3.8%). The influence of
glucosamine-induced insulin resistance became apparent after the
infusion of insulin. Despite the metabolic insulin resistance, the
insulin-induced increments in the amount of farnesylated p21Ras were
significantly (P < 0.005) greater in
glucosamine-treated animals than in saline-treated controls (28 ±
5% vs. 12 ± 3%) compared to preinsulin infusion
levels (Fig. 5A).
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, insulin infusion for 60 min significantly (P < 0.05)
increased the amount of farnesylated p21Ras in the skeletal muscle of
wild-type mice. In contrast, in GFA-overexpressing transgenic mice
(basal insulinemia, 39 ± 2 vs. 16.7 ± 2 µU/ml
in control mice), the basal amount of farnesylated p21Ras was
significantly (P < 0.05) greater than that in control
wild-type mice, and insulin infusion no longer influenced the amount of
farnesylated p21Ras.
Amount of farnesylated p21Ras in transgenic animals with metabolic
insulin resistance
In these sets of experiments, we measured the amount of
farnesylated p21Ras in transgenic animals with well characterized
metabolic insulin resistance: 50% reduction in
IRS-1+/-, IR/IRS-1+/-
mice (23, 24), and A-ZIP/F-1 fatless mice (25). The
IRS-1+/- mice (basal insulinemia ranged from
40–180 µU/ml) had significantly (P < 0.05)
increased amounts of farnesylated p21Ras compared with wild-type mice
(Fig. 6a). Mice that had a concomitant
reduction in the number of IR (IR/IRS-1+/-
double heterozygotes: IR/IRS-1+/-; basal
insulinemia was not different from the IRS-1+/-
mice) displayed normal amounts of farnesylated p21Ras (Fig. 6a).
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). However, by 3 weeks of age
these animals become hyperinsulinemic (249.5 ± 22 vs.
24.4 ± 3 µU/ml in wild-type mice) and insulin resistant and
display significantly (P < 0.05) increased amounts of
farnesylated p21Ras (Fig. 6b).
Effect of hyperinsulinemia on the amount of geranylgeranylated
Rho-A in dogs
As the 
-subunit of FTase is also a component of GGTase I (31),
an enzyme that prenylates Rho proteins, it is conceivable that insulin
also increases the activity of GGTase I. To examine this possibility,
we measured the amount of geranylgeranylated Rho-A in liver samples
from normal dogs. We observed that insulin infusion significantly
increased the amount of geranylgeranylated Rho-A in these animals (Fig. 7). In contrast, the PVD dogs had
increased basal levels of geranylgeranylated Rho-A that were not
significantly increased by insulin infusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These experiments complement our previous findings of increased amounts of farnesylated p21Ras in hyperinsulinemic ob/ob mice and Zucker fa/fa rats (17). However, we have also demonstrated that the amount of farnesylated p21Ras remained normal in denervated rat skeletal muscle (17), a model of insulin resistance not associated with hyperinsulinemia (18). The latter data suggested that hyperinsulinemia and not insulin resistance was responsible for the increased amount of farnesylated p21Ras. Current studies confirm this suggestion by demonstrating that insulin infusions in vivo cause prompt and significant increases in the amount of farnesylated p21Ras in humans, mice, and dogs, thus supporting the causal role of hyperinsulinemia in this process.
We have previously shown that insulin promotes the phosphorylation and activation of the prenyl transferases (8, 9, 10). Increased activity of prenyl transferases parallels the increases in prenylated GTPases (8, 9, 10, 16, 32). Therefore, insulin-induced changes in the amount of prenylated proteins reflect the influence of insulin on the prenyl transferases. The reduction of hyperinsulinemia and concomitant amelioration of insulin resistance in fa/fa Zucker rats by exercise resulted in decreases in the amount of farnesylated p21Ras toward normal levels (17). However, we were unable to correlate the level of insulinemia with the amount of farnesylated p21Ras. Conceivably, the length of time that hyperinsulinemia is present is more important than single measurements of ambient insulinemia. Alternatively, it is possible that the current methodology for measurement of farnesylated p21Ras is not sensitive enough to appreciate minor differences. Finally, one should keep in mind that maximally activated FTase may not be further augmented by even higher concentrations of insulin. This might explain the failure of insulin infusions to further augment already increased levels of farnesylated p21Ras in obese individuals, hyperinsulinemic dogs, and mice overexpressing GFA.
The present results are in agreement with our previous observations
that insulin increases the activity of FTase in tissue culture and in
hyperinsulinemic animals (8, 9, 15, 16, 17). Insulin-induced
phosphorylation of the -subunit of FTase appears to be responsible
for increased FTase enzymatic activity (32). The effects of insulin on
the phosphorylation and activation of FTase appear to be specific and
were not mimicked by IGF-I, EGF, or PDGF (9). Furthermore, we have
shown that inhibition of phosphatidylinositol 3-kinase activity (a key
signaling intermediate of the metabolism aspects of insulin action) did
not alter insulin effects on FTase (32). In contrast, inhibition of the
Ras-mitogen-activated protein kinase pathway completely blocked the
insulin effect on FTase (32), yet did not affect the metabolic aspects
of insulin action.
Even though the increase in the absolute amount of farnesylated p21Ras
is modest (25%), it produces a significantly greater effect
downstream in the signaling cascade. These increases in prenylated
p21Ras result in greater than 2-fold increases in the magnitude of
Ras-GTP loading (15) and the Ras-mediated influence of PDGF on vascular
endothelial growth factor (VEGF) messenger RNA levels (16).
The term insulin resistance generally implies the reduced ability of insulin to promote normal glucose disposal. Other aspects of insulin action may or may not be affected or may be affected to a different degree (3). Several lines of experimental evidence suggest that insulin resistance at the metabolic branch of insulin action is not accompanied by resistance at the mitogenic branch. Recently, Koopmans et al. (4) found that metabolic insulin resistance (diminished glucose uptake) in chronically hyperinsulinemic rats coexisted with increased effects of insulin on de novo lipogenesis. Our current observations strongly suggest that hyperinsulinemia that develops as a compensatory feature of the metabolic insulin resistance increases the activity of the prenyl transferases and the amounts of prenylated p21Ras and Rho-A.
We observed that transgenic animals with insulin resistance at the
level of glucose disposal (such as IRS-1+/-,
A-ZIP/F-1 fatless mice, and GFA-overexpressing mice) have increased
amounts of farnesylated p21Ras in their tissues. Furthermore, an
induction of insulin resistance either by glucosamine infusion (30, 33, 34) or by diverting the pancreatic outflow into the inferior vena cava
(21) was accompanied by significant increments in the amount of
prenylated p21Ras. Experiments with insulin infusions in normal humans,
mice, and dogs indicate that insulin per se is responsible
for the increases in prenylation. Interestingly,
insulin-resistant mice, heterozygous for IRS-1 knockout, do not
display increased levels of farnesylated p21Ras if they have a
concomitant reduction in the number of IRs (i.e. double
heterozygotes, IR/IRS-1+/-), confirming the
requirement for the IR for insulin’s effect on prenylation and that
this effect does not involve the IGF-I receptor (9). Insulin’s
effect on prenylation is mediated exclusively via an intact IR (9).
Thus, insulin failed to stimulate the prenylation of p21Ras in cells
overexpressing either the intact IGF-I receptor or a chimeric
insulin/IGF-I receptor (9). In cells overexpressing IGF-I receptors,
IGF-I had no effect on either phosphorylation of the -subunit of
FTase or the amount of farnesylated p21Ras (9).
We suggest that in the state of insulin resistance at the metabolic
branch of insulin action, the ensuing hyperinsulinemia augments
prenylation of Ras and Rho proteins (Fig. 8). This increase in the magnitude of
insulin action on prenylation, in the presence of its diminished
influence on glucose disposal, is analogous to the pathophysiology of
congenital adrenal hyperplasia; when an enzymatic block in one branch
of adrenal steroidogenesis results in augmentation of the effects of
ACTH on an unaffected branch(s). The precise molecular locus of insulin
resistance is still unknown. However, the inability of insulin to
assure normal glucose uptake and utilization leads to compensatory
hyperinsulinemia that may or may not fully correct glucose disposal.
Because insulin’s ability to stimulate prenyl transferases is not
impaired, hyperinsulinemia increases the amounts of prenylated proteins
in various tissues.
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In summary, our results demonstrate that hyperinsulinemia, either exogenous (infusions of insulin to normal humans, mice, and dogs) or endogenous (insulin resistance in obese humans, PVD dogs, glucosamine-infused rats, IRS-1+/- mice, A-ZIP/F-1 mice, and GFA-overexpressing mice), activates the prenyl transferases in various tissues and increases the amounts of farnesylated p21Ras and geranylgeranylated Rho-A. The ability of insulin to increase the cellular pool of prenylated Ras and Rho proteins may play a critical role in the mechanism of the potentially detrimental influence of hyperinsulinemia.
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Acknowledgments |
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Footnotes |
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Received October 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit. Cell 65:429–434[CrossRef][Medline]
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