(Received for publication, July 31, 1995)
From the
Insulin resistance is a common clinical feature of obesity and
non-insulin-dependent diabetes mellitus, and is characterized by
elevated serum levels of glucose, insulin, and lipids. The mechanism by
which insulin resistance is acquired is unknown. We have previously
demonstrated that upon chronic treatment of fibroblasts with insulin,
conditions that mimic the hyperinsulinemia associated with insulin
resistance, the membrane-associated insulin receptor subunit is
proteolytically cleaved, resulting in the generation of a cytosolic
fragment of the
subunit,
`, and that the generation of
` is inhibited by the thiol protease inhibitor E64 (Knutson, V.
P.(1991) J. Biol. Chem. 266, 15656-15662). In this
report, we demonstrate that in 3T3-L1 adipocytes: 1) cytosolic
`
is generated by chronic insulin administration to the cells, and that
E64 inhibits the production of
`; 2) chronic administration of
insulin to the adipocytes leads to an insulin-resistant state, as
measured by lipogenesis and glycogen synthesis, and E64 totally
prevents the generation of this insulin-induced cellular insulin
resistance; 3) E64 has no effect on the insulin-induced down-regulation
of insulin receptor substrate-1, and therefore insulin resistance is
not mediated by the down-regulation of insulin receptor substrate-1; 4)
under in vitro conditions, partially purified
`
stoichiometrically inhibits the insulin-induced autophosphorylation of
the insulin receptor
subunit; and 5) administration of E64 to
obese Zucker fatty rats improves the insulin resistance of the rats
compared to saline-treated animals. These data indicate that
` is
a mediator of insulin resistance, and the mechanism of action of
`
is the inhibition of the insulin-induced autophosphorylation of the
subunit of the insulin receptor.
Insulin resistance is a characteristic clinical feature of a number of disease states, chief among them diabetes mellitus, and is associated with hyperglycemia, hyperinsulinemia, hyperlipidemia, and hypertension (reviewed in (1) and (2) ). The potential mechanisms by which cellular insulin resistance is generated are many: a mutation in the gene coding for the insulin receptor protein, resulting in a decreased expression of the protein; a decrease in the binding of insulin; a decrease in the number of insulin receptor molecules expressed on the plasma membrane of the target cell; or a so-called ``post-receptor defect'' in which there is a decreased interaction between the insulin receptor and downstream effector molecules.
Natural mutations in the primary sequence of the
insulin receptor have been identified in patients with extreme forms of
insulin resistance (3, 4) . Mutations in the
extracellular ligand binding domain of the receptor have been shown to
result in a decreased affinity of insulin for the
receptor(5, 6, 7, 8, 9) .
Mutations have also been documented in the intracellular subunit
of the receptor, especially in the ATP binding domain and the
autophosphorylation domain of the receptor protein, resulting in a
decreased tyrosine kinase activity of the
receptor(10, 11, 12) . However, of the many
individuals who demonstrate insulin resistance, only a small number of
them have been shown to have mutations in the primary sequence of the
insulin receptor protein (13, 14, 15, 16, 17) .
Therefore, sequence abnormalities in the insulin receptor protein
cannot alone account for insulin resistance, and is not, therefore, a
frequent mechanism leading to the insulin-resistant state (2) .
Insulin resistance induced by a decrease in receptor number could
occur as a result of changes in the mRNA levels for the protein, a
decreased efficiency of post-translational processing of the newly
synthesized receptor protein, or a down-regulation of the level of the
receptor in the cell. A decrease in the cellular levels of insulin
receptor mRNA has been documented in a number of
cases(18, 19, 20, 21, 22) ,
with concomitant decreases in the level of insulin receptor protein. A
decreased rate of insertion of the insulin receptor into the plasma
membrane has also been
demonstrated(10, 11, 12) , primarily as a
result of mutations in the primary sequence of the insulin binding
subunit of the receptor. An elevated rate of degradation of the
insulin receptor protein, resulting in a decreased steady-state level
of cellular receptor, has also been found to be due to specific
modifications in the primary sequence of the
receptor(5, 6) . However, an extremely consistent
finding, both in vivo, in hyperinsulinemic animals models and
humans, and in cultured cells, is that the steady-state level of the
insulin receptor is decreased by chronic exposure of the cells and
tissues to insulin(23, 24, 25, 26) .
In cultured cells, this down-regulation of the insulin receptor has
been demonstrated to be due to an accelerated rate of receptor
inactivation and degradation(27, 28) . Therefore, a
significant degree of insulin resistance could be explained by the
insulin-induced down-regulation of the insulin receptor.
Modifications in the insulin signal transduction pathway distal to
the actual binding of insulin to the receptor could also account for
the insulin-resistant state. These processes could be brought about by
a change in the insulin-induced autophosphorylation of the insulin
receptor subunit, a decrease in the tyrosine kinase activity of
the receptor, or a decrease in the cellular levels of the substrates of
the insulin receptor tyrosine kinase. A significant body of work has
documented that compared to normal patients, in insulin-resistant
patients with non-insulin-dependent diabetes mellitus, insulin binding
to the receptor results in a decreased level of tyrosine
autophosphorylation in the major catalytic domain of the receptor. This
decreased autophosphorylation subsequently leads to the decreased
activation of the receptor tyrosine kinase
activity(29, 30, 31, 32) . Decreased
levels of the coupling molecules or effector molecules in the insulin
signal transduction pathway could also account for insulin resistance.
Multiple substrates of the insulin receptor tyrosine kinase have been
identified(33) . One of the best characterized substrates is
alternately denoted pp160, pp185, or insulin receptor substrate-1
(IRS-1)
(34, 35, 36) . Recent
evidence indicates that upon chronic treatment of cultured cells with
insulin, conditions which result in the down-regulation of the
receptor, the IRS-1 levels in the cell also down-regulate(37) .
These data suggest that insulin resistance might be a consequence of
the insulin-induced down-regulation of coupling molecules in the
insulin signaling pathway.
We have previously demonstrated that upon
chronic treatment of cultured fibroblasts with insulin, the cellular
level of the intact insulin receptor protein decreases, and a fragment
of the insulin receptor subunit is produced and released from the
cellular membranes into the cytosol of the cell(38) . In this
report, we demonstrate that this fragment,
`, is also produced in
cultured adipocytes, and that inhibition of the generation of
`
with the thiol protease inhibitor E64 results in the total abrogation
of insulin-induced insulin resistance in cultured adipocytes, even
though IRS-1 levels are maintained in a down regulated state.
Furthermore, administration of E64 to Zucker fatty rats results in a
decrease of plasma triglyceride levels compared to control animals, and
a decrease in the expression of
` in the tissues of the obese
treated rats. In vitro, partially purified
` inhibits the
insulin-induced autophosphorylation of the insulin receptor. These data
indicate that
` mediates insulin resistance by inhibiting the
autophosphorylation of the insulin receptor.
The pellet was extracted into cell lysis buffer containing 4% Triton
X-100. Following homogenization of the pellet, the sample was incubated
on ice for 1 h to optimize extraction of the membrane proteins, and
then subjected to centrifugation at 200,000 g for 45
min. Following removal of any residual fat cake, this supernatant
contained the extracted membrane proteins, including the intact insulin
receptor.
The cytosolic fraction contained `. This crude
cytosolic fraction was either utilized directly for immunoblot analysis
of
` or IRS-1 (see below) or the
` in this fraction was
further purified by preparative isoelectric focusing. Preparative
isoelectric focusing was performed in the Rotofor apparatus (Bio-Rad),
over a pH range of 2-12. The crude cytosol was added to 2 ml of
40% ampholytes, and the mixture was subjected to focusing in the cooled
chamber, as described by the manufacturer. During the isoelectric
focusing, the temperature was maintained at approximately 5 °C.
Following focusing, the gradient was fractionated into 20 fractions.
The pH of the fractions was determined, and an aliquot of each fraction
was subjected to reducing sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblot analysis to localize
`
(see below). Fractions containing
` were pooled and utilized in
the insulin receptor autophosphorylation assay (see below).
The intact insulin receptor and ` were
immunoprecipitated from the tissue extracts with anti-P5 antibody
coupled to Dynabeads. The Dynabeads were coupled to either anti-P5
antibody or normal rabbit IgG following the manufacturer's
directions. Tissue extracts, either a total of 1 mg of muscle or liver
protein or 0.1 mg of adipose protein, were preabsorbed onto 4 ml of
Dynabeads coupled to rabbit IgG for 5 h, 4 °C with rocking. The
beads were pelleted, and the supernatant was then added to 4 ml of
anti-P5 antibody-coupled beads. The incubation was continued for an
additional 12 h. The beads were pelleted and washed three times with 50
mM Tris, pH 7.4, containing 0.5% deoxycholate and 0.1% sodium
dodecyl sulfate. The proteins absorbed onto the beads were then
subjected to SDS-PAGE and immunoblot analysis as described above,
utilizing the anti-P5 antibody to detect the intact
subunit and
`.
To verify in 3T3-L1 adipocytes the precursor-product
relationship between the insulin receptor subunit and the
cytosolic fragment
`, and to verify that E64, the irreversible
thiol proteinase inhibitor, inhibits the proteolysis of intact
subunit, 3T3-L1 adipocytes were treated with 1.7 µM insulin for 0, 5, or 18 h in the absence or presence of E64.
Immunoblot analysis of the membrane fraction of 3T3-L1 adipocytes with
the anti-P5 antibody (specific for the carboxyl terminus of the
subunit of the insulin receptor) is shown in Fig. 1A,
and densitometric analysis of the blot is shown in Fig. 1B. After 5 h of insulin incubation (-E64, lanes 3 and 4), the intensity of the intact, 92-kDa
subunit of the insulin receptor is decreased by approximately 35%
compared to 0 h of insulin treatment (lanes 1 and 2).
By 18 h of insulin treatment, intact
subunit is even further
decreased by approximately 67% (+E64, lanes 7 and 8). This insulin-induced loss of immunodetectable
subunit is consistent with the insulin-induced loss of insulin
binding activity we have previously reported in 3T3-L1
adipocytes(28) . When 100 µM E64 was added
simultaneously with the insulin, the E64 inhibited the insulin-induced
loss of membrane-associated intact
subunit at 5 h
(+E64, lanes 5 and 6; compare with
control lanes 1 and 2). After coincubation of both
insulin and E64 for 18 h (+E64, lanes 9 and 10), E64 was only partially effective in inhibiting the
insulin-induced down-regulation of the
subunit. Therefore, E64
inhibited the insulin-induced loss of
subunit from the cellular
membranes, but the effectiveness of E64 was lost with the prolonged,
18-h incubation.
Figure 1:
The effect of E64 on
the insulin-induced loss of intact insulin receptor subunit from
membranes of 3T3-L1 adipocytes, and the insulin-induced production of
` in the cellular cytosol. 3T3-L1 adipocytes were treated with 1.7
µM insulin for 0, 5, or 18 h in the presence or absence of
100 µM E64. Intact insulin receptor was extracted from the
total membrane pellet of the cells, and
` was obtained from the
cellular cytosol, as described under ``Experimental
Procedures.'' Panel A, immunoblot analysis of intact
subunit of the insulin receptor. Membrane extracts were from
cells treated with insulin for 0, 5, or 18 h (time of incubation) in
the absence (-E64) or presence (+E64) of
the thiol protease inhibitor. Samples from each experimental condition
were subjected to SDS-PAGE in duplicate. Following SDS-PAGE and
transfer to nitrocellulose, the transfer was incubated with an
anti-peptide antibody directed against the carboxyl terminus of the
subunit of the insulin receptor, as described under
``Experimental Procedures.'' Panel B, densitometric
analysis of the immunoblot in panel A. Solid gray
bars, insulin treatment alone for the indicated incubation time. Cross-hatched bars, insulin plus E64 treatment for the
indicated incubation time. Panel C, immunoblot analysis of
` from the cytosol of treated cells. Cytosol from cells treated
with insulin for 0, 5, or 18 h (incubation time) in the absence
(-E64) or presence (+E64) of E64. Samples
from each experimental condition were subjected to SDS-PAGE in
duplicate. Following SDS-PAGE and transfer to nitrocellulose, the
transfer was incubated with the anti-insulin receptor antibody, as
described under ``Experimental Procedures.'' Panel
D, densitometric analysis of the immunoblot in panel C. Solid gray bars, insulin treatment alone for the indicated
incubation time. Cross-hatched bars, insulin plus E64
treatment for the indicated incubation time. Each sample subjected to
SDS-PAGE, either membrane extract or cytosol, was obtained from
approximately 1
10
cells.
Fig. 1C demonstrates a
representative immunoblot of the effect of insulin and E64 on the
generation of the cytosolic fragment of the insulin receptor
subunit,
`, with densitometric analysis of data from two separate
experiments in Fig. 1D. As demonstrated in lanes 1 and 2, prior to treatment of the cells with insulin, the
level of the 61-kDa
` in the cytosol of the cells is low. However,
after 5 h of insulin treatment (-E64, lanes 3 and 4), the level of
` increased 2-fold above basal,
and after 18 h of insulin treatment of the cells (-E64, lanes 7 and 8), the level of
` was increased
approximately 3.5-fold above basal levels. Simultaneous treatment of
the 3T3-L1 adipocytes with both insulin and E64 resulted in the
inhibition of the production of
` in the cytosol after 5 h of
treatment (+E64, lanes 5 and 6). In
fact, in some experiments, the level of
` detected upon
coincubation with both insulin and E64 was less than the level
of
` found in the control cells. After 18 h of coincubation with
both E64 and insulin (+E64, lanes 9 and 10), elevated levels of
` were demonstrated, again
indicating that the E64 was losing its potency in inhibiting the
insulin-induced production of the fragment. Therefore, E64 prevented
the proteolysis of the insulin receptor
subunit induced by
insulin, and this effect was readily demonstrated after 5 h of
incubation with both insulin and E64. However, the effect of E64 in
3T3-L1 adipocytes was transient, and the cells recovered from the
inhibition by E64 by 18 h.
The data described above indicate that
E64 inhibited the insulin-induced loss of the insulin receptor
subunit from the cellular membranes. Since down-regulation of the
insulin receptor is associated with insulin resistance, it was of
interest to assess the effect of E64 on insulin-induced insulin
resistance in 3T3-L1 adipocytes. Three indices of insulin sensitivity
and insulin resistance were assessed: lipogenesis, glycogen synthesis,
and glucose transport. Fig. 2A demonstrates the effect
of E64 on insulin-stimulated lipogenesis. Consistent with the data
shown with the control (C) cells, we had previously
demonstrated that insulin induces a 3-fold increase in the
incorporation of glucose into lipids
(half-maximal
stimulation at 0.05 nM insulin), and chronic treatment of the
adipocytes with insulin leads to an elevated basal rate of
incorporation, but a loss of insulin-stimulated lipogenesis (9I and 18I). Therefore, chronic treatment of 3T3-L1
adipocytes with insulin resulted in cellular insulin resistance, as
measured by insulin-induced lipogenesis. To assess the effect of E64 on
insulin-induced insulin resistance, the 3T3-L1 adipocytes were
incubated in the presence of insulin alone, or insulin plus E64 for 0,
9, or 18 h. Following exhaustive washing to remove the insulin,
lipogenesis was stimulated with 0, 0.05, and 10 nM insulin.
The results of this experiment are also shown in Fig. 2A. With 9 and 18 h of preincubation with insulin
alone, neither 0.05 nM insulin nor 10 nM insulin
resulted in a rate of glucose incorporation into lipid above the rate
seen with 0 nM insulin. However, after 9 and 18 h, the rate of
basal incorporation was elevated. After 9 h of incubation with both 1.7
µM insulin and 100 µM E64, insulin
sensitivity was readily demonstrated. Interestingly, after 9 h of both
insulin and E64, the basal rate of incorporation was at the basal level
demonstrated in the control cells, and the maximal uptake quantitated
at 10 nM insulin was significantly higher than the maximal
uptake demonstrated in the control cells. Therefore, the incubation of
the 3T3-L1 adipocytes with both insulin and E64 for 9 h not only
preserved the insulin responsiveness of the cells, but, in fact, made
them more responsive than the control cells. After 18 h of incubation
with both insulin and E64, the ``protective'' effects of E64
were lost, and insulin resistance demonstrated by the cells was
comparable to that demonstrated in cells incubated with insulin alone
for 18 h. Cells incubated with E64 alone demonstrated
concentration-response curves similar to control cells (data not
shown).
Figure 2:
Insulin-induced lipogenesis, glycogen
synthesis, and glucose uptake in basal and insulin-resistant 3T3-L1
adipocytes: effects of E64 on insulin resistance. Panel A,
effect of E64 on insulin-induced lipogenesis in cells chronically
pretreated with insulin. 3T3-L1 adipocytes were pretreated with 1.7
µM insulin alone for 0 (C), 9 (9I), or
18 h (18I), or with insulin plus 100 µM E64 for 9 (9EI) or 18 h (18EI). Following exhaustive washing to
remove any residual insulin, the cells were further stimulated with 0
nM insulin (black bars), 0.05 nM insulin (gray bars), or 10 nM insulin (cross-hatched
bars) before quantitating lipogenesis as described under
``Experimental Procedures.'' Under basal conditions in the
control cells, glucose incorporation into lipids denoted as 1250
``cpm glucose incorporated into lipid'' is equivalent to a
rate of 13.5 nmol/min/10 cells. Panel B, effect of
E64 on insulin-induced glycogen synthesis in cells chronically
pretreated with insulin. 3T3-L1 adipocytes were pretreated with 1.7
µM insulin alone for 0 (C), 9 (9I), or
18 h (18I), or with insulin plus 100 µM E64 for 9 (9EI) or 18 h (18EI). Following exhaustive washing to
remove any residual insulin, the cells were further stimulated with 0
nM insulin (black bars), 5 nM insulin (gray bars), or 25 nM insulin (cross-hatched
bars) before quantitating glycogen synthesis as described under
``Experimental Procedures.'' Panel C, effect of E64
on insulin-induced glucose uptake in cells chronically pretreated with
insulin. 3T3-L1 adipocytes were pretreated with 1.7 µM
insulin alone for 0 (C), 9 (9I), or 18 h (18I), or with insulin plus 100 µM E64 for 9 (9EI) or 18 h (18EI). Following exhaustive washing to
remove any residual insulin, the cells were further stimulated with 0
nM insulin (black bars), 2.5 nM insulin (gray bars), or 25 nM insulin (cross-hatched bars) before quantitating glucose
uptake as described under ``Experimental
Procedures.''
The effect of E64 on glycogen synthesis was also explored.
We had previously demonstrated that insulin stimulation of 3T3-L1
adipocytes induces a robust 14-fold induction of glycogen synthesis,
with half-maximal induction occurring at approximately 5 nM insulin. As with lipogenesis, chronic insulin
pretreatment of the cells resulted in an increase in basal glycogen
synthesis, but a total loss of insulin-inducible glycogen
synthesis.
Chronic insulin pretreatment induces insulin
resistance of glycogen synthesis. These results are recapitulated in Fig. 2(panel B), demonstrating a 14-fold induction in
control (C) cells and insulin resistance in insulin-pretreated
cells (9I and 18I). The effect of E64 on glycogen
synthesis was then determined, and data on the coincubation of the
cells with both insulin and E64 for 9 or 18 h are also shown in Fig. 2B. The inclusion of 100 mM E64 in the 9-
and 18-h insulin incubation (9IE and 18IE) resulted
in an improvement of insulin sensitivity of the cells, compared to
cells incubated with insulin alone. After both 9 and 18 h of
preincubation with both insulin and E64, acute insulin stimulation
could induce a 2-fold increase in glycogen synthesis. Unlike the
results with lipogenesis, E64 treatment did not decrease the basal
rates of glycogen synthesis to control cell levels (compare C (0 nM insulin) with 9EI (0 nM insulin)
and 18EI (0 nM insulin)), and the maximal rates of
glycogen synthesis in the E64-treated cells were not as high as the
rates demonstrated in the control cells (compare C (25 nM insulin) with 9EI (25 nM insulin) and 18EI (25 nM insulin)). However, insulin responsiveness was
clearly apparent in the E64-treated cells but absent in the cells not
treated with E64 (compare 9I (25 nM insulin) with 9EI (25 nM insulin) or 18I (25 nM insulin) with 18EI (25 nM insulin)). Therefore,
incubation of the 3T3-L1 adipocytes with both insulin and E64 preserved
the insulin responsiveness of the cells.
The effect of E64 on glucose uptake was also pursued. We had previously demonstrated that insulin stimulates glucose uptake into 3T3-L1 adipocytes 8-fold, and that chronic insulin treatment of the cells induces an insulin-resistant state by elevating basal glucose uptake with a concomitant reduction in insulin stimulated uptake to approximately 2-fold. These data are recapitulated in Fig. 2C, where control cells (C) demonstrate an 8-fold induction of glucose transport, with half-maximal uptake demonstrated at 2.5 nM insulin, and cells treated with insulin for 9 (9I) or 18 (18I) h demonstrate elevated basal rates of uptake and very little additional insulin-induced uptake. The effects of E64 on glucose uptake were then determined, and these data are also presented in Fig. 2C. In marked contrast to the data presented in panels A and B, incubation of the adipocytes with E64 did not preserve the insulin-responsiveness of the glucose transport process in these cells. After 9 h of insulin pretreatment, basal transport was elevated approximately 4-fold above the control cells, and subsequent insulin challenge induced only a 1.3-fold increase in transport. The inclusion of E64 with the insulin incubation had no additional effect on the glucose transport profile (compare 9EI with 9I). Similar results were obtained after an 18-h incubation with insulin alone or insulin plus E64.
The data presented in Fig. 2demonstrate that desensitization of the different signaling pathways modulated by insulin respond in different ways to treatment of the cells with E64.
The findings presented
above with lipogenesis and glycogen synthesis are consistent with the
interpretation that ` is mediating the insulin resistance in
3T3-L1 adipocytes. When the fragment is present, insulin resistance is
demonstrated. When the production of the fragment is inhibited, insulin
resistance is not demonstrated. However, earlier reports had
demonstrated that IRS-1 is down-regulated by insulin treatment of
3T3-L1 adipocytes(37) . As an early coupling protein in the
insulin signal transduction pathway, down-regulation of IRS-1 could
inhibit the propagation of signal down the insulin-induced cascade, and
lead to insulin resistance. It is also possible that E64 might inhibit
the down-regulation of IRS-1. To test this, cytosol was isolated from
adipocytes preincubated in the presence of insulin and/or E64.
Following SDS-PAGE and transfer to nitrocellulose, the transfer was
incubated with antibody directed against IRS-1. The immunoblot results
are shown in Fig. 3A, and quantitation of the blot is
presented in Fig. 3B. In the cytosol from control cells (lanes 1 and 2), IRS-1 was detected at a mass of 160
kDa, consistent with previous reports(37) . Treatment of the
adipocytes for 5 h with insulin resulted in a 50% decrease in
immunodetectable IRS-1 (lanes 3 and 4), and exposure
of the intact cells to insulin for 18 h further down-regulated the
level of IRS-1 to approximately 20% of control levels (lanes 7 and 8). The addition of E64 to the insulin incubation had
no effect on the level of expression of IRS-1. After 5 h of incubation
with both insulin and E64 (lanes 5 and 6), IRS-1 was
down-regulated to the same degree demonstrated in the presence of
insulin alone, and after 18 h of incubation with both insulin and E64 (lanes 9 and 10), no detectable change in the level
of IRS-1 could be observed compared to the level of IRS-1 seen with
insulin alone. Therefore, in contrast to the effect of E64 on the
production of
`, E64 had no effect on the insulin-induced
down-regulation of IRS-1. Therefore, the insulin resistance induced by
hyperinsulinemia in 3T3-L1 adipocytes is not mediated by a decrease in
the level of IRS-1.
Figure 3:
The effect of E64 on the insulin-induced
down-regulation of IRS-1. 3T3-L1 adipocytes were treated with 1.7
µM insulin for 0, 5, or 18 h in the presence or absence of
100 µM E64. Cellular cytosol was prepared and subjected to
SDS-PAGE and immunoblot analysis with anti-IRS-1 antibody, as described
under ``Experimental Procedures.'' Panel A,
immunoblot analysis of IRS-1 in cytosol. Cytosol from cells treated
with insulin for 0, 5, or 18 h (incubation time) in the absence
(-E64) or presence (+E64) of the thiol
protease inhibitor. Samples from each experimental condition were
subjected to SDS-PAGE in duplicate. Panel B, densitometric
analysis of the immunoblot in panel A. Solid gray
bars, insulin treatment alone for the indicated incubation time. Cross-hatched bars, insulin plus E64 treatment for the
indicated incubation time. Each sample subjected to SDS-PAGE was
obtained from approximately 1 10
cells.
It has been demonstrated that the
insulin-induced autophosphorylation of the insulin receptor is reduced
in tissues from insulin-resistant patients, compared to the
insulin-induced autophosphorylation of the receptor in normal
individuals(29, 30, 31, 32) . We
proceeded to ascertain if, under in vitro conditions, `
was able to inhibit the insulin-induced autophosphorylation of the
intact
subunit of the insulin receptor. Intact insulin receptor
was partially purified from untreated, insulin-free 3T3-L1 adipocytes.
` was partially purified from the cytosol of 3T3-L1 adipocytes
that had been treated for 18 h with 1.7 µM insulin. A
constant amount of intact receptor (7 µl) was incubated with
varying amounts of
` (0-140 µl). The relative amounts of
intact receptor and
` utilized in this experiment were determined
so that the amount of intact
subunit present in 7 µl of
detergent extract was equivalent, by immunoblot analysis, to the amount
of
` present in 75 µl of purified cytosol. After mixing and
incubation with or without insulin and
[
-
P]ATP, the samples were subjected to
SDS-PAGE and scanning. Quantitation of the radioactivity present in the
92 kDa band is shown in Fig. 4. Panel A demonstrates
the effect of
` on basal and insulin-stimulated incorporation. In
the absence of
`, insulin induced the incorporation of
P into the 92-kDa
subunit of the insulin receptor.
The presence of
` has no effect on the basal incorporation (minus
insulin) of
P into the
subunit at any concentration
of
`. However, as the concentration of
` was increased,
insulin-stimulated phosphorylation of the
subunit was decreased.
As shown in Panel B, the stimulation of incorporation induced
by insulin in the absence of
` was 2.5-fold. As the concentration
of
` was increased, insulin-induced stimulation of incorporation
was inhibited. Interpolation of the data shown in Fig. 4B indicates that the insulin-induced phosphorylation of
subunit was totally inhibited at a
`:
ratio of approximately
1.
Figure 4:
The effect of partially purified ` on
insulin-stimulated receptor autophosphorylation. A constant
concentration of intact insulin receptor was incubated with graded
concentrations of partially purified
`. Following incubation in
the presence or absence of insulin, [
P]ATP was
added to the incubation to induce the autophosphorylation of the
receptor. The samples were then subjected to SDS-PAGE and radiometric
scanning to quantitate the radioactivity associated with the insulin
receptor
subunit at 92 kDa, as described under
``Experimental Procedures.'' Panel A,
autophosphorylation of the intact
subunit as a function of the
relative ratio of
` to
subunit. The value of
`/
was quantitated as described under ``Experimental
Procedures.'' Black bars, incubation in the presence of
insulin. Gray bars, incubation in the absence of insulin. Panel B, -fold stimulation of insulin receptor
autophosphorylation as a function of the ratio of
` to
.
-Fold stimulation of insulin receptor autophosphorylation was
calculated as (dpm incorporated with insulin)/(dpm incorporated without
insulin). A value of unity indicates no insulin-stimulated
autophosphorylation of the insulin receptor
subunit.
Interestingly, [P]phosphate was also added
to a protein band migrating with a mass of 61 kDa, corresponding to
`. Quantitation of the radioactivity in the
` 61-kDa band is
shown in Fig. 5. Phosphorylation of
` occurred to an equal
extent in the absence or presence of stimulation with insulin, and the
level of incorporation increased as a linear function of the amount of
added
`. Phosphorylation of
` also occurred in the absence of
intact insulin receptor (data not shown, manuscript in preparation).
Therefore, from the data presented in Fig. 4and Fig. 5,
under in vitro conditions,
` stoichiometrically inhibited
the insulin-induced phosphorylation of the
subunit of the intact
insulin receptor. In addition,
` was itself phosphorylated.
Figure 5:
Phosphorylation of `, the cytosolic
fragment of the insulin receptor
subunit. The insulin receptor
autophosphorylation assay in the presence of
` was performed as
described in the legend to Fig. 4and under ``Experimental
Procedures.'' From the same SDS-PAGE gels that resulted in the
data presented in Fig. 4, the radioactive band migrating at 61
kDa,
`, was quantitated for radioactivity.
, assay
incubation in the presence of 100 nM insulin;
, assay
incubation in the absence of insulin.
The
data presented in Fig. 1and Fig. 2suggest that E64
inhibits cellular insulin resistance by inhibiting the production of
the fragment of the insulin receptor subunit,
`. To assess
the effect of E64 on the insulin-resistant state manifest in the
complex system of a whole animal, we utilized the obese,
insulin-resistant Zucker fatty rat. This rodent model has been utilized
as a model for insulin resistance and Type 2 non-insulin-dependent
diabetes mellitus(42, 43, 44) . The animals,
both obese and age-matched lean Zucker rats, were injected with E64 on
a daily basis for 3 months. The protocol was initiated at an age when
the insulin-resistant state (determined by serum triglyceride levels)
was fully expressed(42, 45) . Serum insulin, glucose,
and triglycerides were measured weekly. Consistent with earlier
reports, we found the obese rats to be normoglycemic (185 mg/dl in both
the fatty and lean rats) but hyperinsulinemic (40 microunits/ml in the
obese animals, and 7 microunits/ml in the lean animals), compared to
the non-obese animals. No statistically significant changes in serum
glucose and insulin levels were noted over the course of the protocol
in comparing the treated and control animals (data not shown). However,
the obese animals had elevated blood levels of triglycerides compared
to the lean rats. Lean rats injected with E64 demonstrated no change in
blood levels of triglycerides compared to saline-injected controls
(data not shown). Weights of neither the obese nor the lean rats was
changed by E64 compared to the saline controls (data not shown). The
effect of daily injection of E64 on serum triglyceride levels is shown
in Fig. 6. In the 2-week period preceding the beginning of the
E64 administration, the blood levels of triglycerides were comparable
in both the test and control group of animals. Over the next 3 months
of the protocol, the blood levels of triglycerides in the E64 group
were maintained at the pretreatment levels. However, the obese rats
injected with saline demonstrated a steady increase in blood
triglyceride levels over the 3-month protocol. Linear regression
analysis of the triglyceride levels in E64-treated rats demonstrated an
increase in blood levels of only 0.62 mg/dl/day, while the saline
control animals increased blood triglyceride levels at a rate of 7.34
mg/dl/day. Therefore, E64 inhibited the rate of increase in blood
triglycerides by a factor of 11.8. While the injection of E64 did not
reverse the insulin-resistant state manifest by the rodents at the
onset of the protocol, it prevented the worsening of the
insulin-resistant state.
Figure 6:
The effect of E64 on blood triglyceride
levels in Zucker fatty rats. Female Zucker fatty rats were injected on
a daily basis with E64. Blood was drawn on the indicated days and the
blood levels of triglycerides were quantitated, as described under
``Experimental Procedures.'' , saline-injected controls;
, animals injected with E64. *, p < 0.075; **, p < 0.020.
To determine the effect of E64 treatment on
the level of intact insulin receptor and ` in the tissues of the
rodents, liver, skeletal muscle, and adipose tissue were excised from
both lean and obese animals, extracted with detergent-containing
buffers, and the extracts were preabsorbed with normal rabbit IgG and
then immunoprecipitated with anti-P5 antibody prior to immunoblot
analysis with anti-P5 antibody. The intensity of the bands at 92 kDa
(intact
subunit) were determined, and the results are presented
in Fig. 7. All samples were analyzedsimultaneously. Therefore,
direct comparisons between samples are possible. It should also be
noted that adipose tissue has a relatively high receptor level.
Consequently, in order to maintain all of the samples within the linear
range of detection, 0.1 mg of adipose total protein was subjected to
immunoprecipitation compared to 1 mg of liver or muscle protein. In the
lean rats (L), E64 treatment had no effect on the level of
intact insulin receptor
subunit in any tissue. A comparison of
the level of the
subunit in the untreated lean animals (L/Saline) and the untreated obese (F/Saline) rats
indicated that in all three tissues,
subunit levels are
substantially down regulated in the obese rats. Total
subunit
level is decreased by 4-fold in the untreated liver of the obese rats,
and comparable decreases are demonstrated in muscle and adipose tissue.
E64 treatment of the obese rats dramatically reversed the
down-regulation of the levels of
subunit (compare F/Saline with F/E64). With all three tissues, E64 treatment of the
animals resulted in increased levels of intact
subunit such that
the
subunit levels in the fatty rats treated with E64 were
comparable to the levels of
subunit present in the lean rats.
Thus, the data presented in Fig. 7are consistent with the
interpretation that E64 inhibits the insulin-induced proteolysis of the
intact
subunit of the insulin receptor, thereby elevating the
level of receptor in the tissues of these animals, improving their
insulin resistance.
Figure 7:
The effect of E64 on the level of the
subunit of the insulin receptor in tissues from Zucker rats. Upon
completion of the protocol described in the text and Fig. 6,
tissue was excised from the animals, extracted into
detergent-containing buffer, and the level of the intact
subunit
of the insulin receptor was quantitated, as described under
``Experimental Procedures.'' In each immunoprecipitation, 1
mg of extracted protein from liver and muscle was utilized, and 0.1 mg
of extracted protein from adipose tissue was utilized. F/Saline, fatty Zucker rats injected with saline alone; F/E64, fatty Zucker rats injected with E64; L/Saline,
lean Zucker rats injected with saline alone; L/E64, lean
Zucker rats injected with E64. Black bars, liver; gray
bars, muscle; cross-hatched bars, adipose
tissue.
The effect of E64 treatment on the level of
` in the tissues of the obese rats is shown in Fig. 8. As
described above for the quantitation of the intact
subunit, 0.1
mg of adipose protein was subjected to immunoprecipitation compared to
1 mg of liver, muscle, kidney, or spleen protein. In the obese rats,
E64 treatment resulted in decreased levels of
` in muscle, adipose
tissue, and kidney by factors of 1.9, 2.2, and 1.4, respectively.
Spleen, a tissue not normally viewed as an insulin target tissue,
demonstrated no E64-induced change in
`. Paradoxically, liver from
E64-treated rats demonstrated an elevated level of
`. We currently
have no explanation for this finding. A low level of
` could be
detected in the tissues of the lean rats, and E64 treatment had no
effect on the level of expression of
` in the tissues of these
animals (data not shown).
Figure 8:
The
effect of E64 on the level of ` production in tissues from obese
Zucker rats. Upon completion of the protocol described in the text and Fig. 6, tissue was excised from the animals and extracted into
detergent-free buffer, and the level of
` was quantitated as
described under ``Experimental Procedures.'' In each
immunoprecipitation, 1 mg of extracted protein from liver, muscle,
kidney, and spleen was utilized, and 0.1 mg of extracted protein from
adipose tissue was utilized.
We had previously demonstrated that in fibroblasts, chronic
insulin treatment induces the proteolytic release of a fragment of the
subunit of the insulin receptor from the cellular membranes into
the cytosol. This earlier study also demonstrated that the thiol
protease inhibitor E64 inhibited this insulin-induced
activity(38) . The studies reported here in 3T3-L1 adipocytes
are consistent with our earlier findings in fibroblasts; the 61-kDa
fragment of the insulin receptor is released from the cell membranes by
chronic treatment with insulin into the cytosol of the cells. The
studies in this report extend our earlier findings by ascribing
functional significance to this fragment of the insulin receptor,
denoted
`, and proposing a mechanism of action.
Upon chronic
treatment of 3T3-L1 adipocytes with insulin, the normally robust
lipogenic response of the cells to insulin is eliminated. This insulin
resistance is overcome when E64 is administered to the cells
concomitantly with the desensitizing dose of insulin. At 9 h of
simultaneous incubation with both insulin and E64, identical conditions
to those which inhibit the production of `, insulin resistance is
eliminated. However, if the incubation with both E64 and insulin is
continued for 18 h, the cells overcome the effects of E64 and
`
production is again demonstrated and insulin resistance is seen. These
data provide a strong correlation between insulin resistance and
`; when
` is present, the cells are insulin-resistant, but
when
` is reduced or absent, the cells are insulin-sensitive. The
same correlation is demonstrated with an alternate index of insulin
sensitivity, glycogen synthesis. Insulin-induced glycogen synthesis is
absent from the 3T3-L1 adipocytes when the cells are pretreated for
5-18 h with insulin alone, but under conditions that inhibit the
production of
` (simultaneous treatment with E64), insulin
sensitivity is retained. There is, however, a significant difference
between the effect of E64 on lipogenesis versus glycogen
synthesis. With lipogenesis, E64 maintains the basal level of
lipogenesis at levels comparable to the level seen in the control
cells. In contrast, with glycogen synthesis, cells treated with both
E64 and insulin demonstrate an elevated rate of basal incorporation
similar to that seen in cell treated with insulin alone. An even
greater discrepancy exists in the effects of E64 on the insulin-induced
desensitization of glucose transport. E64 had no effect in these cells
on the recovery of desensitized glucose uptake. The basis for these
differences is unknown, but possibly reflects differences in the
insulin-induced signaling pathways for lipogenesis, glycogen synthesis,
and glucose transport. Subtle differences in the pathways for
lipogenesis and glycogen synthesis may exist, but more dramatic
differences between the signaling pathway leading to glucose transport versus lipogenesis are apparent.
It has recently been
demonstrated that chronic treatment of 3T3-L1 adipocytes with insulin
results in the down-regulation of IRS-1. Since IRS-1 is a substrate for
the insulin receptor tyrosine kinase, it is reasonable to suggest that
a decrease in the cellular concentration of IRS-1 below a critical
level could create a rate-limiting step in the insulin signal
transduction cascade, resulting in insulin resistance. If E64 inhibited
the down-regulation of IRS-1, then the correlation between insulin
resistance and E64 treatment could be explained on the basis of the
action of E64 on IRS-1 as readily as the effect of E64 on `.
However, the data of Fig. 3clearly demonstrate that E64 has no
effect on the insulin-induced down-regulation of IRS-1. Under
conditions of concomitant insulin and E64 treatment, where insulin
responsiveness is maintained, the level of IRS-1 is down-regulated to
the same degree as in the presence of insulin alone. Therefore,
down-regulation of IRS-1 does not contribute to the insulin-induced
insulin resistance of 3T3-L1 adipocytes when utilizing lipogenesis and
glycogen synthesis as indices of insulin action. However, glucose
transport, like IRS-1 levels, are not affected by the E64, suggesting
that IRS-1 levels become limiting in the induction of glucose transport
in the desensitized cells.
A mechanism to support the correlation
between E64, `, and insulin resistance is provided by the data of Fig. 4and Fig. 5. Coincubation of intact insulin receptor
with partially purified
` results in the inhibition of the
insulin-induced autophosphorylation of the intact
subunit. When
the ratio of
subunit to
` is unity, total inhibition of
phosphorylation is demonstrated. In the intact 3T3-L1 adipocytes, total
insulin resistance was found after 9 h of incubation with insulin. We
have previously demonstrated that after 5-6 h of incubation of
these cells with insulin, the receptor level declines by approximately
50%(28) . As the receptor down-regulates,
` is generated,
such that by 5-6 h after insulin addition, the concentration of
intact
subunit in the cellular membranes should be approximately
equal to the concentration of
` in the cytosol. Therefore,
sufficient quantities of
` are generated in the 3T3-L1 adipocytes
by 9 h to account for the complete insulin resistance demonstrated in Fig. 2.
A major problem with the interpretation of the data
of Fig. 4and Fig. 5relates to the purity of `.
While preparative isoelectric focusing decreases the number of
contaminating proteins in the preparation,
` is not the only
protein in the preparation (data not shown).
Therefore, it
is possible that
` itself is not inhibiting the
autophosphorylation of the insulin receptor but that another factor in
the preparation, which is induced by insulin treatment and inhibited by
E64 treatment, could be affecting receptor autophosphorylation.
However, our preliminary data indicate that cytosol isolated from
control cells or cells incubated with both insulin and E64 does not
have any effect on insulin receptor autophosphorylation (data not
shown).
Resolution of this question will be possible when
we have purified
` to homogeneity. Our current efforts are
directed in this area.
` is itself phosphorylated, but in an
insulin-independent manner. This finding is consistent with results
generated with deletion mutants of the insulin receptor. In these
studies, insulin receptor cDNA sequences coding for the extracellular
domain of the receptor were deleted, resulting in the expression of a
membrane-anchored (46) or fully soluble (47, 48, 49) cytoplasmic domain of the
insulin receptor. This protein was found to be a constitutively active
tyrosine kinase, contributing to the conclusion that the insulin
binding domain of the insulin receptor acts in a negative regulatory
manner, inhibiting the tyrosine kinase activity of the
subunit(46) . Studies are currently under way in our laboratory
to ascertain if
` has tyrosine kinase activity. If it does have
this activity, it would present a potential new mechanism of insulin
resistance;
` not only inhibits the insulin receptor
autophosphorylation, but if
` has tyrosine kinase activity, it may
inappropriately phosphorylate substrates, resulting in aberrant insulin
signaling.
The studies cited above on the expression of the
cytoplasmic domain of the insulin receptor were apparently not extended
to determine the effect of this domain on the signal transduction
capacity of the endogenous insulin receptor. However, studies of this
nature have been performed with the -adrenergic
receptor(41) . The third cytoplasmic loop of the
adrenergic receptor has been expressed as a soluble protein in
cells along with the parent
adrenergic receptor. The
expression of the cytoplasmic domain has been found to inhibit the
ability of the parent receptor to activate phospholipase C. Therefore,
this study on the
adrenergic receptor forms a precedent for our
finding that a fragment of the cytosolic domain of the insulin receptor
inhibits receptor function. However, in this study on the adrenergic
receptor, the cytosolic fragment of the receptor was expressed as a
result of transfection with modified DNA; the fragment is not a protein
normally expressed in the wild-type cell. In the study we report here,
the fragment of the insulin receptor is expressed in wild-type cells,
in response to ``pathological'' concentrations of insulin,
resulting in cellular insulin resistance.
To determine the effect of
E64 on insulin resistance under in vivo conditions, Zucker
fatty rats were injected with E64 on a daily basis. As shown in Fig. 6, E64 has an immediate effect on serum triglyceride
levels, maintaining the triglyceride levels in the treated rats at
pretreatment levels. This was in marked contrast to the saline-treated
fatty rats, where the serum triglyceride levels continued to increase.
Therefore, injection of E64 prevented the worsening of hyperlipidemia
in this animal model. The data presented in Fig. 7demonstrate
that concomitant with an E64-induced improvement in the insulin
resistance of the obese rats, the level of the subunit in insulin
target tissues recovered from a down-regulated state. These data
substantiate the interpretation that E64 prevents the insulin-induced
degradation of the insulin receptor. Fig. 8demonstrates the
effect of the E64 protocol on
` production in tissues from obese
Zucker rats. In muscle, adipose tissue, and kidney, E64 decreased
` levels approximately 2-fold. These data are consistent with the
view that E64 blocks
` production, leading to the observed
improvement of the insulin-resistant state shown in Fig. 6.
It is important to note that this animal protocol was initiated when the insulin-resistant state was fully manifest in these rats. Additional studies are in progress to ascertain if hyperlipidemia can be prevented in the Zucker fatty rats by administration of E64 to the animals earlier in their lives, before the onset of florid insulin resistance. A significant drawback to the use of Zucker fatty rats to study insulin resistance is that, unlike insulin resistance in humans, the Zucker fatty rats are normoglycemic. Therefore, glycemic control cannot be utilized as an index of the degree of insulin resistance. Future animal studies on the use of E64 will include other animal models of insulin resistance, where changes in blood glucose and insulin can be utilized as indices of the effectiveness of E64 in decreasing insulin resistance.
In summary, this report demonstrates
a causal link between the production of `, a cytosolic fragment of
the insulin receptor, and the generation of insulin resistance. Insulin
treatment of cultured adipocytes results in a time-dependent loss of
intact
subunit from the cellular membranes, and a concomitant
generation of
` in the cell cytosol, in which the production of
` is inhibited by the thiol protease inhibitor E64. While insulin
treatment of the adipocytes also leads to insulin resistance, as
measured by the loss of insulin-stimulated lipogenesis, glycogen
synthesis, and glucose uptake, concurrent treatment of the adipocytes
with insulin and E64 inhibits the generation of the insulin-resistant
glycogen synthesis and lipogenesis (but not glucose uptake) under
conditions that also inhibit the generation of
`. E64 has no
effect on the insulin-induced down-regulation of IRS-1, so insulin
resistance cannot be correlated to the down-regulation of IRS-1 in
3T3-L1 adipocytes. In in vitro experiments, partially purified
` inhibits the insulin-induced autophosphorylation of the
subunit of the intact insulin receptor, such that complete inhibition
of insulininduced autophosphorylation is achieved when the molar ratio
of
` to
subunit is unity. This suggests that
` induces
insulin resistance in intact cells by inhibiting the
autophosphorylation of the insulin receptor. Finally, in intact
animals, E64 improves the insulin-resistant state of Zucker fatty rats,
lowering the blood levels of triglycerides in this rat model of insulin
resistance. In this animal model, administration of E64 both increases
the level of intact
subunit and decreases the level of
` in
the animal tissues, when compared to the corresponding tissues from
saline-treated animals. Therefore, these data indicate that insulin
resistance is mediated by a cytosolic fragment of the insulin receptor,
`, which is generated by hyperinsulinemia.