From the Endocrinology Section, Carl T. Hayden
Veterans Administration (VA) Medical Center, Phoenix, Arizona 85012, the ¶ Research Service, VA Medical Center, Omaha, Nebraska 68105, the
Division of Diabetes, Endocrinology and Metabolism,
University of Nebraska Medical Center, Omaha, Nebraska 68198, and the
** Molecular and Cellular Biology Program, Arizona State University,
Tempe, Arizona 85287
Received for publication, August 31, 2000, and in revised form, December 7, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In adult animals, the major effect of
insulin on protein turnover is inhibition of protein degradation.
Cellular protein degradation is under the control of multiple systems,
including lysosomes, proteasomes, calpains, and giant protease. Insulin
has been shown to alter proteasome activity in vitro and
in vivo. We examined the inhibition of protein degradation
by insulin and insulin analogues (LysB28,ProB29-insulin (LysPro),
AspB10-insulin (B10), and
GluB4,GlnB16,PheB17-insulin (EQF))
in H4, HepG2, and L6 cells. These effects were compared with receptor
binding. Protein degradation was examined by release of trichloroacetic
acid-soluble radioactivity from cells previously labeled with
[3H]leucine. Short- and intermediate-lived proteins were
examined. H4 cells bound insulin with an EC50 of 4.6 × 10 The major effect of insulin on whole body protein turnover is
inhibition of protein degradation (1). Insulin also stimulates the
synthesis of selected proteins and overall protein synthesis under
certain conditions such as growth and development (2, 3). In the adult
animal, however, total protein synthesis is not increased by insulin,
and the protein anabolic effect of the hormone is actually an
anti-catabolic property, i.e. inhibition of degradation
(1).
The mechanisms of cellular protein degradation and the control of these
processes are poorly understood. Currently, the major degradative
systems are considered to be the lysosome, the proteasome, and various
cytoplasmic and cellular proteases such as the calpain family and a
recently described giant protease (4-7), which may functionally
overlap proteasome activities. In general, lysosomes appear responsible
for degradative processing of most endocytosed proteins and for
autophagy under extreme catabolic states (4). The proteasome is a
multifunctional organelle with multiple forms, e.g. 20 S and
26 S, which degrades abnormal and targeted proteins (ubiquitin,
ATP-dependent pathway) and has various specific functions (antigen processing, etc.) (5). Calpains participate in
Ca2+-mediated degradative activity (6). Various other
proteases have specialized or uncertain roles but have a relatively
minor contribution to cellular protein balance.
Overall, cellular protein degradation can also be divided according to
the half-lives of the proteins. This is particularly relevant, because
many of the techniques for assessing protein turnover involve
prelabeling with radioactive amino acids. The duration of labeling,
chase, and subsequent release can provide data on turnover of different
classes of proteins (e.g. short-lived versus
long-lived).
Protein degradation is altered by many factors, including hormones,
amino acid levels, metabolic substrates (e.g. glucose), ions, and others (8-14). In addition, tissues such as liver and muscle
may interact in regulating degradation in the alternate tissue; for
example, amino acids from muscle breakdown may alter hepatic protein
degradation. With this degree of complexity and interaction, it is not
surprising that the specific effects of insulin on protein degradation
and the mechanisms of those effects are not well understood. We
(15-17) and others (18) have shown that insulin alters proteasomal
activity in broken cell preparations and in intact cells. In recent
work, we have shown effects of insulin on ubiquitin-mediated protein
degradation (19). The present studies examine the inhibition of protein
degradation by insulin and various insulin analogues in three different
cell types under different conditions. We have compared these effects to the binding of the different peptides.
Materials--
The rat hepatoma cell line, H4-II-E, the human
hepatoma cell line, HepG2, and the rat skeletal muscle cell line, L6,
were purchased from the ATCC (Rockville, MD). Culture media were from Sigma (St. Louis, MO), fetal calf serum
(FCS)1 and gentamicin were
from Life Technologies Inc. (Gaithersburg, MD). Biosynthetic human
insulin, LysB28,ProB29-insulin (LysPro),
AspB10-insulin (B10), and
GluB4,GlnB16,PheB17-insulin (EQF)
and 125I-insulin (labeled in the A14 tyrosine position)
were gifts of Dr. R. Chance (Lilly Research Laboratories).
[3H]Leucine was from Amersham Pharmacia Biotech
(Piscataway, NJ). All other chemicals were of at least reagent grade.
Cell Culture--
L6 myoblasts were plated in 24-well culture
dishes at a density of 7000 cells/cm2. Cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
10% FCS and 10 µg/ml gentamicin and incubated at 37 °C in an
atmosphere of 5% CO2/95% air. The medium was changed every 2-3 days. L6 myoblasts spontaneously differentiate upon confluency and fuse to form multinucleated myotubes (20). Cells are
used when differentiated (10 days after plating).
H4 cells and HepG2 cells were grown in 24-well plates (starting
density = 3.4 × 104 cells/cm2). The
growth medium consisted of Eagle's minimum essential medium (MEM) with
10% FCS and 10 µg/ml gentamicin. Cells were incubated at 37 °C in
an atmosphere of 5%CO2/95% air. The medium was changed every 2-3 days, and the cells were used when confluent (~5 days).
Binding Assay--
Binding of 125I-insulin to cells
was carried out at 4 °C for 3 h. Radioactivity (10-20,000 cpm)
was added with either insulin or analogues in serum-free medium
containing 0.1% BSA and 2 mM TES, pH 7.5. After incubation
unbound material was removed and the cells were washed twice in PBS, pH
7.4. Solubilized cells were counted in a gamma-counter.
Intermediate-lived Protein Degradation--
Protein degradation
was measured as described by Gunn et al. (21) for liver
cells with slight modifications. For L6 cells, on day 9 after plating,
the growth medium was removed from the cells and replaced with
leucine-free DMEM containing 10% FCS and 10 µg/ml gentamicin and 1 µCi/ml [3H]leucine. Cells were incubated for 18 h
to allow labeling of cellular proteins with [3H]leucine.
Labeling medium was replaced with incubation/chase medium (DMEM
containing 2 mM unlabeled leucine, 20 mM TES,
pH 7.5, and 0.1% BSA) and incubated for 3 h at 37 °C (chase
time). The 3-h chase period allowed for the removal of short-lived
proteins. Following this the chase medium was removed and the cells
were rinsed in chase medium. Insulin or analogues (10 Short-lived Protein Degradation--
The effect of insulin on
short-lived protein degradation was investigated essentially as
described for intermediate-lived protein degradation except there was
no chase period before the addition of insulin or analogues. Following
labeling for 18 h with radioactive leucine, cells were rinsed
twice with incubation/chase medium, insulin or analogues were added,
and the cells incubated at 37 °C for 3 h.
Statistical Analysis--
Comparisons between the different
conditions were by analysis of variance with Dunnett's multiple
comparison post test. p < 0.05 was taken as significant.
H4-II-E Hepatoma cells--
Because insulin effects on cellular
function are initiated by receptor binding, the binding of
[125I]iodoinsulin and the effects of native insulin and
various analogues were examined. Fig. 1
shows the competitive displacement of [125I]iodoinsulin
by native insulin and three analogues, LysPro, B10, and EQF. Insulin
and LysPro are similar. B10 has a 2-fold increased affinity and EQF a
15-fold reduced affinity as compared with insulin. The EC50
values are shown in Table I.
Fig. 2A shows that H4 cells
are very sensitive to the inhibition of protein degradation by insulin
(Table II), as has also been seen for
other actions of insulin (22). LysPro and B10 are even more effective
(p < 0.001) (Table II). Despite the significant differences in binding, EQF (Fig. 2C) had an effect similar
to insulin on protein degradation (Table II). The studies shown in Fig.
2 were done with overnight labeling and a 3-h incubation with insulin
or analogues in complete medium as described under "Experimental
Procedures." Several aspects of the results are notable, including
the very high sensitivity to insulin and the differences in activity of
the analogues relative to their binding potencies as compared with
insulin.
The studies were repeated using a modification of the experimental
protocol. In the approach shown in Fig. 2 the degradation of both
short- and intermediate-lived proteins is measured. To remove the
contribution of the rapidly turning over proteins and to focus on
proteins with longer half-lives, a 3-h chase period was added, and the
incubation with insulin or analogues was extended to 4 h (Fig.
3). Under these conditions the
curves are shifted to the right, indicating lesser sensitivity for
insulin and LysPro (Fig. 3A). Despite this, LysPro remains
more effective than insulin (Table II). B10 (Fig. 3B) is
significantly more effective than insulin under these conditions (Table
II). EQF (Fig. 3C) remains equivalent to insulin (Table II).
Thus, discrepancies between binding and activity for LysPro and EQF,
but not for B10, remain. For this reason, studies were done in a
different cell line.
HepG2 Cultured Hepatocytes--
Fig.
4 shows competitive displacement of
[125I]iodoinsulin by insulin and analogues from HepG2
cells. The pattern is similar to H4 cells with insulin and LysPro being
equivalent and with B10 having an increased and EQF a marked decrease
in apparent affinity (Table I).
Fig. 5 compares the effectiveness of the
analogues with insulin on the inhibition of short-lived protein
degradation in HepG2 cells. Insulin, LysPro (Fig. 5A), and
B10 (Fig. 5B) are very similar (Table II). EQF (Fig.
5C) is much less effective (p < 0.01).
To examine intermediate-lived proteins, a 3-h chase and a 4-h
incubation were done (Fig. 6). In these
studies, the dose-response curve for EQF was different from insulin
(Fig. 6C), and the EC50 was significantly higher
(Table II). Insulin, LysPro, and B10 were equivalent.
L6 Cells--
Fig. 7 shows binding
of 125I-insulin to L6 myotubes and displacement by insulin
and analogues. The EC50 values are shown in Table I.
Although the affinities followed the same relative order as with other
cells, the absolute differences were less and no statistical significance was seen among the materials. Protein degradation was
examined for short-lived (Fig. 8) and
intermediate-lived (Fig. 9) proteins.
Figs. 8A and 9A show insulin and LysPro to
be equal, Fig. 9B shows an increased potency of B10, and EQF
is less potent (Figs. 8C and 9C) (Table II).
Although the effect of insulin to inhibit protein degradation is
well established in both in vivo and in vitro
studies, the mechanisms by which the hormone has this effect and the
proteolytic pathways involved are unclear. Protein degradation is a
complex set of processes involving multiple pathways and multiple
classes of proteins with different turnover rates. Insulin has variable effects on the different processes. The present series of studies demonstrates this complexity. We compared the effects of insulin on
protein degradation under different conditions with several insulin
analogues in various cell types. The rapidly absorbed analogue LysPro
(23) was examined along with two other analogues that have altered
binding and processing characteristics. B10 has an increased binding
affinity (24), but reduced susceptibility to cellular processing and
degradation (25). The B10 position is an early cleavage site for the
degradation of insulin (26) and the Asp for His substitution alters
cellular processing in several ways, including slower receptor
dissociation and intracellular processing (25). EQF has several
substitutions that decrease both binding and susceptibility to
degradation (27). The B16 site, which is altered in EQF insulin, is one
of the earliest cleavage sites in insulin (26). This study confirms the
increased binding affinity of B10 (24), the reduced receptor binding of EQF (27), and the unaltered binding of LysPro. Similar results were
seen with H4 hepatocytes, HepG2 hepatocytes, and L6 myotubes.
These studies illustrate the complexities in cellular protein
degradation and the effects of insulin. The results varied with study
conditions and cell type. H4 cells were very sensitive to insulin.
Under conditions that measured the turnover of both short- and
intermediate-lived proteins, an extremely sensitive insulin effect was
seen (EC50 = 4.2 × 10 The biological effects of insulin and analogues on protein degradation
relative to binding are shown in Table
III. No consistent relationship between
binding and biological activity on protein degradation by the analogues
was seen. This is in contrast to the high correlation between insulin
receptor binding and glucose transport (29) and lipogenesis (30-34).
This is true for the analogues used here (29, 31, 33-35) and other
insulin analogues (29-35). We also found that glucose incorporation
into glycogen in L6 cells and H4 cells correlate with receptor
binding.2
9 M. LysPro was similar. The affinity of
B10 was increased 2-fold; that of EQF decreased 15-fold. Protein
degradation inhibition in H4 cells was highly sensitive to insulin
(EC50 = 4.2 × 10
11 and 1.6 × 10
10 M, short- and intermediate-lived protein
degradation, respectively) and analogues. Despite similar binding,
LysPro was 11- to 18-fold more potent than insulin at inhibiting
protein degradation. Conversely, although EQF showed lower binding to
H4 cells than insulin, its action was similar. The relative binding
potencies of analogues in HepG2 cells were similar to those in H4
cells. Examination of protein degradation showed insulin, LysPro, and
B10 were equivalent while EQF was less potent. L6 cells showed no
difference in the binding of the analogues compared with insulin, but
their effect on protein degradation was similar to that seen in HepG2
cells except B10 inhibited intermediate-lived protein degradation
better than insulin. These studies illustrate the complexities of
cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The
differences seen in the action of the analogues cannot be attributed to
binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12
to 10
6 M), in incubation medium, were added
to the cells and incubated for 4 h at 37 °C (incubation time).
For some experiments DMEM, which normally contains 20 mM
glucose, was replaced with DMEM containing 5 mM glucose
(low glucose medium). The incubation was stopped by placing the cells
on ice and adding an equal volume of 6 M acetic acid
containing 2% Triton X-100 to solubilize the cells. Aliquots of the
cell/medium mix were analyzed for protein degradation by precipitation
in 10% (final concentration) trichloroacetic acid. Protein degradation
was taken as the percent trichloroacetic acid-soluble radioactivity.
Assays in hepatoma cells were carried out as described for L6 cells
except the cell labeling medium was leucine-free MEM with 10% FCS, 10 µg/ml gentamicin, and 1 µCi/ml leucine. The incubation/chase medium
consisted of MEM with 20 mM TES, pH 7.5, 0.1% BSA, and 2 mM leucine. All incubations were identical to the muscle cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Binding of 125I-insulin to H4
cells. Cells were incubated for 3 h at 4 °C with 10,000 cpm 125I-insulin with and without unlabeled insulin or
analogues. Binding was determined as described under "Experimental
Procedures." The data show the mean of three individual experiments
carried out in triplicate.
Displacement of [125I] iodoinsulin binding by insulin and
analogues
View larger version (15K):
[in a new window]
Fig. 2.
Degradation of short-lived proteins in H4
cells. The dose-response to insulin and the analogues LysPro
(A), B10 (B), and EQF (C) are shown.
Cells were labeled overnight with [3H]leucine. Cells were
then washed, and medium containing excess unlabeled leucine was added
with and without insulin or analogues. Cells were incubated for 3 h at 37 °C. Protein degradation was determined as described under
"Experimental Procedures." Cells degraded 15.2 ± 0.6%/3 h of
protein in the absence of insulin or analogues. Data show mean ± S.E. of 3-12 experiments.
The effect of insulin and analogues on protein degradation
View larger version (16K):
[in a new window]
Fig. 3.
Degradation of intermediate-lived proteins in
H4 cells. The dose-response to insulin and the analogues LysPro
(A), B10 (B), and EQF (C) are shown.
Cells were labeled overnight with [3H]leucine. Cells were
then chased for 3 h in medium containing excess unlabeled leucine.
The chase medium was removed, and medium containing 2 mM
leucine was added with and without insulin or analogues. Cells were
incubated for 4 h at 37 °C. Protein degradation was determined
as described under "Experimental Procedures." Cells degraded
18.5 ± 0.8%/4 h of protein in the absence of insulin or
analogues. Data show mean ± S.E. of four to six
experiments.
View larger version (19K):
[in a new window]
Fig. 4.
Binding of 125I-insulin to HepG2
cells. Cells were incubated for 3 h at 4 °C with 10,000 cpm 125I-insulin with and without unlabeled insulin or
analogues. Binding was determined as described under "Experimental
Procedures." The data show the mean ± S.E. of four to five
individual experiments carried out in triplicate.
View larger version (14K):
[in a new window]
Fig. 5.
Degradation of short-lived proteins in HepG2
cells. The dose response to insulin and the analogues LysPro
(A), B10 (B), and EQF (C) are shown.
The experiments were carried out as described in Fig. 2. Cells degraded
17.4 ± 0.7%/3 h of protein in the absence of insulin or
analogues. Data show mean ± S.E. of four to five
experiments.
View larger version (14K):
[in a new window]
Fig. 6.
Degradation of intermediate lived proteins in
HepG2 cells. The dose response to insulin and the analogues LysPro
(A), B10 (B), and EQF (C) are shown.
The experiments were carried out as described in Fig. 3. Cells degraded
18.2 ± 0.8%/4 h of protein in the absence of insulin or
analogues. Data show mean ± S.E. of three to four
experiments.
View larger version (18K):
[in a new window]
Fig. 7.
Binding of 125I-insulin to L6
cells. Cells were incubated for 3 h at 4 °C with 20,000 cpm 125I-insulin with and without unlabeled insulin or
analogues. Binding was determined as described under "Experimental
Procedures." The data show the mean ± S.E. of three to seven
individual experiments carried out in triplicate.
View larger version (14K):
[in a new window]
Fig. 8.
Degradation of short-lived proteins in L6
cells. The dose response to insulin and the analogues LysPro, B10,
and EQF are shown. The experiments were carried out as described in
Fig. 2. Cells degraded 25.4 ± 0.2%/3 h of protein in the absence
of insulin or analogues. Data show the mean ± S.E. of three
experiments.
View larger version (14K):
[in a new window]
Fig. 9.
Degradation of intermediate-lived proteins in
L6 cells. The dose response to insulin and the analogues LysPro
(A), B10 (B), and EQF (C) are shown.
The experiments were carried out as described in Fig. 3. Cells degraded
21.9 ± 0.4%/4 h of protein in the absence of insulin or
analogues. Data show mean ± S.E. of 3 to 14 experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11 M).
When a preincubation was added to eliminate rapidly turning over
proteins, the EC50 increased ~4-fold (1.6 × 10
10 M) but remained very sensitive (22). The
binding affinity is typical (4.6 × 10
9
M) (28). This shows a dissociation between binding and activity.
Potency of insulin analogues to inhibit protein degradation relative to
binding as compared to insulin
Of equal note is that the results varied in different cell types and with different experimental conditions. In general, H4 cells responded better to the analogues than to insulin, and HepG2 cells responded less well to the analogues. Relative to binding, LysPro was 13-21 times more effective than insulin in H4 cells but only half as effective in HepG2 cells. EQF was up to 22 times more effective in H4 cells but less than half as effective in HepG2 cells. The simple conclusion is that differences in action of insulin analogues on protein degradation cannot be attributed solely to their receptor binding.
These results illustrate the importance of factors beyond receptor binding in the actions of insulin. LysPro insulin binds to the insulin receptor with an affinity identical to insulin (Ref. 23 and our results). The effects of this analogue on glucose metabolism are identical to insulin (34, 36). Other effects, including cell growth and mitogenesis, are also similar to insulin (27, 34). B10 has an increased affinity for the insulin receptor and correspondingly greater effects on glucose metabolism and on mitogenesis (29, 34). The latter effect has been attributed to increased IGF-I receptor binding (29).
In the present study LysPro was more potent than insulin in decreasing protein degradation in H4 cells but not HepG2 or L6 cells (Table II). B10 had a greater effect than insulin in H4 cells but not in HepG2 cells despite the increased binding of B10 in HepG2 cells. EQF had a markedly decreased binding affinity in both hepatic cell lines but had effects similar to insulin on protein degradation in H4 cells. In HepG2 cells EQF was less potent than insulin at inhibiting protein degradation. Overall, there was a very poor correlation between binding and inhibition, which extended across all cell types for all analogues.
Previous studies have shown that biological activity (glucose transport and metabolism, cell growth, and mitogenesis) and insulin receptor binding activity correspond closely for insulin and most analogues (24, 27, 29-35). In the present study of insulin effects on protein degradation, these correlations break down. The activities of the analogues relative to binding vary among different cell types and with different assay conditions (Table III). In H4 cells all of the analogues tended to have increased activity relative to binding, when compared with insulin. EQF had a considerably greater effect than insulin on intermediate-lived proteins relative to binding. In contrast, in HepG2 cells, another cultured hepatocyte line, the analogues tended to have less activity relative to binding, as compared with insulin. Again there were differences between effects on intermediate- and short-lived proteins. EQF was much less effective on short-lived protein degradation and B10 less effective on intermediate lived proteins. LysPro was very similar to insulin. Lastly, in L6 cells, EQF was less effective than insulin on short-lived protein degradation but equally effective when intermediate protein degradation was measured. LysPro and B10 were similar to insulin when activity relative to binding was considered. This lack of correlation demonstrates that post-receptor binding events must be involved.
The conclusion that the control of protein degradation by insulin
requires more than just receptor binding is supported by previous
literature. Draznin's group (37) and our laboratory (38) reported that
cellular insulin processing and degradation were required for the
effect of insulin on protein degradation. We have shown direct effects
of insulin on isolated proteasomal activity (15, 16, 19), raising the
possibility that intracellular insulin or insulin degradation products
(39) may play a role in mediating some of the effects of insulin on
protein degradation. In studies of cellular processing of the analogues
used in this study, the correlation between cellular processing and
effect on protein degradation is much greater than that between binding and activity.3 The present
study has shown that the effects of insulin and various insulin
analogues on protein degradation vary significantly in different cell
types and with different experimental conditions. These differences
cannot be explained by alterations in receptor binding. Post-receptor
mechanisms, including intracellular processing and degradation, deserve evaluation.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Endocrinology (CS/111E), Veterans Administration Medical Center, 650 E. Indian School Rd., Phoenix, AZ 85012. Tel.: 602-277-5551 (ext. 6690); Fax: 602-200-6004; E-mail: janet.fawcett@med.va.gov.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M007988200
2 J. Fawcett, F. G. Hamel, R. G. Bennett, Z. Vajo, and W. C. Duckworth, unpublished observation.
3 J. Fawcett, F. G. Hamel, R. G. Bennett, Z. Vajo, and W. C. Duckworth, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FCS, fetal calf serum; LysPro, LysB28- ProB29-insulin; B10, AspB10-insulin; EQF, GluB4,GlnB16,PheB17-insulin; DMEM, Dulbecco's modified Eagle's medium; MEM, Eagle's minimal essential medium; BSA, bovine serum albumin; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Rooyackers, O. E., and Nair, K. S. (1997) Annu. Rev. Nutr. 17, 457-485[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Kimball, S. R.,
Horetsky, R. L.,
and Jefferson, L. S.
(1998)
Am. J. Physiol.
274,
C221-C228 |
3. | Thompson, M. G., and Palmer, R. M. (1998) Cell. Signal. 10, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
4. | Bohley, P., and Seglen, P. O. (1992) Experientia 48, 151-157[Medline] [Order article via Infotrieve] |
5. | Coux, O., Tanaka, K., and Goldberg, A. L. (1996) Annu. Rev. Biochem. 65, 801-847[CrossRef][Medline] [Order article via Infotrieve] |
6. | Sorimachi, H., Ishiura, S., and Suzuki, K. (1997) Biochem. J. 328, 721-732[Medline] [Order article via Infotrieve] |
7. | Yao, T., and Cohen, R. E. (1999) Curr. Biol. 9, R551-R553[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Beynon, R. J.,
and Bond, J. S.
(1986)
Am. J. Physiol.
251,
C141-C152 |
9. | Mortimore, G. E. (1982) Nutr. Rev. 40, 1-12 |
10. | Mortimore, G. E., Poso, A. R., and Lardeux, B. R. (1989) Diabetes Metab. Rev. 5, 49-70[Medline] [Order article via Infotrieve] |
11. | Garlick, P. J., McNurlan, M. A., Bark, T., Lang, C. H., and Gelato, M. C. (1998) J. Nutr. 128, 356S-359S[Medline] [Order article via Infotrieve] |
12. | Fulks, R. M., Li, J. B., and Goldberg, A. L. (1975) J. Biol. Chem. 250, 290-298[Abstract] |
13. | Kettelhut, I. C., Wing, S. S., and Goldberg, A. L. (1988) Diabetes/Metabolism Rev. 4, 751-772[Medline] [Order article via Infotrieve] |
14. |
Mitch, W. E.,
and Goldberg, A. L.
(1996)
N. Engl. J. Med.
335,
1897-1905 |
15. | Bennett, R. G., Hamel, F. G., and Duckworth, W. C. (1997) Diabetes 46, 197-203[Abstract] |
16. |
Duckworth, W. C.,
Bennett, R. G.,
and Hamel, F. G.
(1994)
J. Biol. Chem.
269,
24575-24580 |
17. | Hamel, F. G., Bennett, R. G., Harmon, K. S., and Duckworth, W. C. (1997) Biochem. Biophys. Res. Commun. 234, 671-674[CrossRef][Medline] [Order article via Infotrieve] |
18. | Li, B. G., Fang, C. H., and Hasselgren, P. (2000) Int. J. Biochem. Cell Biol. 32, 677-686[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Bennett, R. G.,
Hamel, F. G.,
and Duckworth, W. C.
(2000)
Endocrinology
141,
2508-2517 |
20. | Mandel, J.-L., and Pearson, M. L. (1974) Nature 251, 618-620[Medline] [Order article via Infotrieve] |
21. | Gunn, J. M., Clark, M. G., Knowles, S. E., Hopgood, M. F., and Ballard, F. J. (1977) Nature 266, 58-60[Medline] [Order article via Infotrieve] |
22. | Kelley, D. S., Becker, J. E., and Potter, V. (1978) Cancer Res. 38, 4591-4600[Medline] [Order article via Infotrieve] |
23. |
Holleman, F.,
and Hoekstra, J. B. L.
(1997)
N. Eng. J. Med.
337,
176-183 |
24. | Slieker, L. J., Brooke, G. S., DiMarchi, R. D., Flora, D. B., Green, L. K., Hoffmann, J. A., Long, H. B., Fan, L., Shields, J. E., Sundell, K. L., Surface, P. L., and Chance, R. E. (1997) Diabetologia 40, S54-S61[CrossRef][Medline] [Order article via Infotrieve] |
25. | Hamel, F. G., Siford, G. L., Fawcett, J., Chance, R. E., Frank, B. H., and Duckworth, W. C. (1999) Metabolism 48, 611-617[Medline] [Order article via Infotrieve] |
26. |
Duckworth, W. C.,
Bennett, R. G.,
and Hamel, F. G.
(1998)
Endocrine Rev.
19,
608-624 |
27. | Slieker, L. J., Brooke, G. S., Chance, R. E., Fan, L., Hoffmann, J. A., Howey, D. C., Long, H. B., Mayer, J., Shields, J. E., Sundell, K. L., and DiMarchi, R. D. (1994) in Current Directions in Insulin-like Growth Factor Research (LeRoith, D. , and Raizada, M. K., eds), Vol. 343 , pp. 25-32, Plenum Press, New York |
28. | Ballard, F. J., Wong, S. S., Knowles, S. E., Partridge, N. C., Martin, T. J., Wood, C. M., and Gunn, J. M. (1980) J. Cell. Physiol. 105, 335-346[Medline] [Order article via Infotrieve] |
29. | Hansen, B. F., Danielsen, G. M., Drejer, K., Sørensen, A. R., Wiberg, F. C., Klein, H. H., and Lundemose, A. G. (1996) Biochem. J. 315, 271-279[Medline] [Order article via Infotrieve] |
30. | Chu, Y. C., Zong, L., Burke, G. T., and Katsoyannis, P. G. (1992) J. Protein Chem. 11, 571-577[Medline] [Order article via Infotrieve] |
31. | Burke, G. T., Hu, S. Q., Ohta, N., Schwartz, G. P., Zong, L., and Katsoyannis, P. G. (1990) Biochem. Biophys. Res. Commun. 173, 982-987[Medline] [Order article via Infotrieve] |
32. | Svoboda, I., Brandenburg, D., Barth, T., Gattner, H. G., Jirácek, J., Velek, J., Bláha, I., Ubik, K., Kasicka, V., Pospísek, J., and Hrbas, P. (1994) Biol. Chem. Hoppe-Seyler 375, 373-378[Medline] [Order article via Infotrieve] |
33. | Vølund, A., Brange, J., Drejer, K., Jensen, I., Markussen, J., Ribel, U., Sørensen, A. R., and Schlichtkrull, J. (1991) Diabet. Med. 8, 839-847[Medline] [Order article via Infotrieve] |
34. | Kurtzhals, P., Schäffer, L., Sørensen, A., Kristensen, C., Jonassen, I., Schmid, C., and Trüb, T. (2000) Diabetes 49, 999-1005[Abstract] |
35. | Drejer, K. (1992) Diabetes Metab. Rev. 8, 259-285[Medline] [Order article via Infotrieve] |
36. | Howey, D. C., Bowsher, R. R., Brunelle, R. L., and Woodworth, J. R. (1994) Diabetes 43, 396-402[Abstract] |
37. |
Draznin, B.,
and Trowbridge, M.
(1982)
J. Biol. Chem.
257,
11988-11993 |
38. | Peavy, D. E., Edmondson, J. W., and Duckworth, W. C. (1984) Endocrinology 114, 753-760[Abstract] |
39. | Hamel, F. G., Fawcett, J., and Duckworth, W. C. (2000) Diabetes 49, A26 (abstr.) |