(Received for publication, June 20, 1995; and in revised form, November 6, 1995)
From the
Over short time periods glucose controls insulin biosynthesis predominantly through effects on preexisting mRNA. However, the mechanisms underlying the translational control of insulin synthesis are unknown. The present study was carried out to determine the effect of glucose on the activity and/or phosphorylation status of eukaryotic initiation and elongation factors in islets. Glucose was found to increase the activity of the guanine nucleotide-exchange factor eIF-2B over a rapid time course (within 15 min) and over the same range of glucose concentrations as those that stimulate insulin synthesis (3-20 mM). A nonmetabolizable analogue of glucose (mannoheptulose), which does not stimulate insulin synthesis, failed to activate eIF-2B.
The best characterized mechanism for modulating
eIF-2B activity involves changes in the phosphorylation of the
-subunit of its substrate eIF-2. However, in islets, no change in
eIF-2
phosphorylation was seen under conditions where eIF-2B
activity was increased, implying that glucose regulates eIF-2B via an
alternative pathway.
Glucose also did not affect the phosphorylation states of three other regulatory translation factors. These are the cap-binding factor eIF-4E, 4E-binding protein-1, and elongation factor eEF-2, which do not therefore seem likely to be involved in modulating the translation of the preproinsulin mRNA under these conditions.
Glucose is the major physiological stimulus for insulin
biosynthesis in pancreatic cells with control occurring
principally at the level of translation of preformed mRNA (Permutt and
Kipnis, 1972; Permutt, 1974; Itoh and Okamoto, 1980; Itoh et
al., 1982; Welsh et al., 1986). The mechanism(s) by which
elevated concentrations of glucose stimulate insulin biosynthesis is
not known, although it is clear that the effect is exerted primarily at
the level of peptide chain initiation (Permutt, 1974). The effect is
selective for proinsulin synthesis, in that the stimulation of
proinsulin synthesis by glucose is proportionally much greater than
that of overall protein synthesis (Permutt and Kipnis, 1972).
The translational machinery in eukaryotes is highly complex and involves a number of translation initiation and elongation factors. Changes in the activities of several translation factors are believed to play roles in the regulation of translation in animal cells (Hershey, 1991; Proud, 1992; Redpath and Proud, 1994). Two factors in particular have been studied in detail.
eIF-2 mediates the binding of the initiator
Met-tRNA to the 40 S subunit of the ribosome, and changes in its
activity are important in regulating translation under a variety of
conditions. The best characterized mechanism involves changes in the
phosphorylation of its -subunit at Ser-51. eIF-2(
P)
competitively inhibits the factor (eIF-2B) which recycles eIF-2 between
successive rounds of initiation (Hershey, 1991; Rowlands et
al., 1988a). This recycling process involves the exchange of GDP
bound to eIF-2 for GTP by eIF-2B, which regenerates active
eIF-2
GTP. It plays an important role in the control of
translation (Price and Proud, 1994) and the activity of eIF-2B is
potentially subject to regulation by a variety of mechanisms including
eIF-2
phosphorylation, allosteric control, and phosphorylation of
eIF-2B on its largest (
) subunit. At least three protein kinases
have been shown to phosphorylate this polypeptide and evidence has been
presented that all three may modulate eIF-2B activity: casein kinases-1
and -2 to activate and glycogen synthase kinase-3 to inhibit (Dholakia
and Wahba, 1988; Welsh and Proud, 1993; Denslow et al., 1994;
Singh et al., 1994). (
)
Phosphorylation of
eIF-4E, which binds to the 5`-cap of the mRNA, correlates positively
with rates of translation, and evidence has been provided that this
factor plays a key role in the control of the translation of mRNAs
whose 5`-untranslated regions are rich in secondary structure, which
impedes mRNA translation (Hershey, 1991; Proud, 1992; Kozak, 1992).
eIF-4E is now known to be regulated by an additional mechanism,
involving an inhibitory protein (4E-BP1 = eIF-4E
binding-protein-1), ()which undergoes phosphorylation in
response to certain agents that stimulate translation, and, apparently
as a consequence, dissociates from eIF-4E (Pause et al., 1994;
Haystead et al., 1994; Lin et al., 1994). This is
thought to lead to activation of eIF-4E, perhaps by allowing it to bind
to other components of the ``cap-binding complex,'' which
include eIF-4A, a bidirectional RNA helicase.
The elongation step of translation is also subject to regulation, in this case through the phosphorylation of elongation factor-2 (eEF-2). eEF-2 mediates the translocation step of peptide-chain elongation: phosphorylation of eEF-2 by the Ca/calmodulin-dependent eEF-2 kinase causes complete inactivation of eEF-2 (Redpath et al., 1993; Redpath and Proud, 1994).
This work was undertaken to examine the effects of
elevated glucose levels on the activity and/or phosphorylation of the
regulatory translation factors mentioned above. In this study we show
that glucose rapidly activates eIF-2B independently of changes in the
phosphorylation of eIF-2, suggesting direct regulation of this
factor. Glucose also does not alter the phosphorylation states of
eIF-4E or eEF-2 in islets, or the association of 4E-BP1 with eIF-4E.
For measurement of
total protein synthesis, 200 islets were preincubated in 100 µl of
Hanks' balanced salt solution containing 3 or 20 mM glucose for 40 min. This medium was then removed and replaced with
100 µl of Hanks' balanced salt solution containing 150
µCi of [S]methionine. Labeling was
terminated after 20 min by the addition of ice-cold Hanks'
balanced salt solution. Measurement of counts incorporated into total
protein was carried out essentially as described by Guest et
al. (1989).
Figure 1:
Effect of glucose on proinsulin
biosynthesis in isolated rat islets of Langerhans. Rat islets of
Langerhans were incubated for 20 min in medium containing 3 mM (lane 1) or 20 mM (lane 2) glucose and
[S]methionine. Newly synthesized proinsulin was
then immunoprecipitated and analyzed by Tricine/SDS-PAGE and
fluorography. The position of molecular mass markers in kilodaltons is
indicated. This result is typical of those obtained from five separate
experiments.
Figure 2: Effect of glucose on eIF-2B activity in isolated rat islets of Langerhans. Panel A, rat islets of Langerhans incubated in medium containing 20 mMD-glucose for the indicated periods of time. The data represent the mean of three determinations of eIF-2B activity in a single experiment, where the standard errors were less than 5% for each value. This result is typical of that obtained from five separate time course experiments. Panel B, effect of various D-glucose concentrations on eIF-2B activity in isolated rat islets of Langerhans. Islets were incubated for 30 min in medium containing the indicated D-glucose concentrations. The data represent the mean of three determinations in a single experiment, where the standard errors were less than 5% for each value. This result is typical of those obtained from five separate experiments in which the maximal extent of activation ranged from 230 to 370% of the control (3 mM glucose).
The response was assessed over a range of glucose concentrations (3-20 mMD-glucose, 30 min treatment). eIF-2B activity was increased in a dose-dependent fashion (Fig. 2B). This dose response closely matches that reported elsewhere for the effects of varied glucose concentrations on insulin synthesis (Guest et al., 1989).
In a typical experiment where 20 mM glucose resulted in an increase in eIF-2B activity (282% of control where glucose was 3 mM) the hexokinase inhibitor mannoheptulose had no effect on eIF-2B activity in islets on its own (98% of control). However, when used in combination with 20 mM glucose, it suppressed the activation of of eIF-2B (151% of control). This is an accordance with the previously reported inhibition by mannoheptulose of the increase in L-type pyruvate kinase mRNA in response to glucose (Marie et al., 1993).
Figure 3:
Time courses of the effect of glucose on
the phosphorylation of eIF-2, eIF-4E, and eEF-2 (in isolated rat
islet cells). Rat islets of Langerhans were incubated in 20 mMD-glucose for 0 (lane 1), 10 (lane 2),
20 (lane 3), 30 (land 4), and 60 min (lane
5). Cell extracts were subject one-dimensional isoelectric
focusing and Immunoblotted using an anti-eIF-2
(Panel A),
anti-eIF-4E (Panel B) antibody. Detection was by enhanced
chemiluminescence. The data shown are representative of four individual
experiments. Labeled arrowheads indicate the positions of the
unphosphorylated and phosphorylated forms of eIF-2
in Panel A and of eIF-4E in Panel B.
We also analyzed the levels of phosphorylation of eIF-4E and eEF-2 in extracts from cells treated with low or high glucose concentrations. No change was seen in the ratio of unphosphorylated to phosphorylated protein following treatment with 20 mMD-glucose for eIF-4E (Fig. 3B) or eEF-2 (data not shown). For eIF-4E, densitometric analysis of blots revealed that approximately 50% of protein was in the phosphorylated form in both control and glucose-treated cells.
Figure 4:
Association of eIF-4E with 4E-BP1 in
extracts from islets. Islets were incubated in 3 mMD-glucose (lane 1) or with 20 mMD-glucose for 15 (lane 2) or 30 min (lane
3), and then extracts were prepared. eIF-4E and associated
proteins were isolated by affinity binding to
mGTP-Sepharose and analyzed by SDS-PAGE followed by
immunoblotting. Blots were visualized by enhanced chemiluminescence.
The positions of eIF-4E and eIF-4EBP1 are
marked.
In the present study we show that the treatment of islets
with glucose results in an increase in eIF-2B activity. This could
account for the rise in total protein synthesis observed when islets
are exposed to raised external glucose concentrations, since enhanced
eIF-2B activity should result in increased supply of Met-tRNA to the
ribosome and this is required for the translation of all mRNAs.
Modulation of eIF-2B, and thus eIF-2 activity, is important in the
overall control of translation under specific conditions such as heme
deficiency in reticulocytes and in the presence of double-stranded RNA
in reticulocytes and other cell types. Since the rise in in eIF-2B
activity was, if anything, larger than the increase in total protein
synthesis, components other than eIF-2B are presumably limiting. The
rise in proinsulin synthesis is much larger than that in total protein
synthesis. Lomedico and Saunders(1977) suggested that this might
reflect the operation of the ``Lodish model.'' Lodish(1974)
proposed, on the basis of his studies with the mRNAs for the - and
-chains of hemoglobin, that mRNAs differ in their intrinsic
efficiency, such that when the activities of certain components of the
translational machinery were limiting, inefficient mRNAs would be
translated much less readily than efficient ones. Activation of those
limiting components would, of course, lead to an overall stimulation of
translation, but would allow especially marked increases in the
translation of the inefficient mRNAs. Lomedico and Saunders(1977)
postulated that insulin was an example of an inefficient mRNA and might
be regulated in this way. Alternatively, there may be a
``directed'' mechanism operating to secure the increased
translation specifically of this mRNA involving specific features of
this mRNA or proteins interacting with it.
Although the activity of
eIF-2B was increased, no change in the level of phosphorylation of
eIF-2 was seen, indicating that the rise in its activity was not
due, as one might have expected, to a decrease in eIF-2
phosphorylation. It should also be noted that the assays performed here
contain a large excess of ``added'' substrate eIF-2 relative
to the endogenous eIF-2 in the sample under study: under this condition
effects of eIF-2(
P) (a competitive inhibitor) (Rowlands et
al., 1988a) derived from the extract should be minimized if not
eliminated, as discussed previously (Rowlands et al., 1988b;
Welsh and Proud, 1992). This suggests that glucose enhances the
activity of eIF-2B by mechanism distinct from eIF-2
phosphorylation. There are now known to be a number of situations where
eIF-2B activity is altered in intact cells or tissues without any
detectable change in eIF-2
phosphorylation (Kimball and Jefferson,
1988; Rowlands et al., 1988b; Jeffrey et al., 1990;
Welsh and Proud, 1992). (
)
In the absence of a change in
eIF-2 phosphorylation, how can the changes in eIF-2B activity be
brought about? It is likely that they involve direct regulation of
eIF-2B itself, and this might in principle be a consequence of either
allosteric regulation or of covalent modification. Several agents, such
as nicotinamide adenine nucleotides and polyamines, modulate eIF-2B
activity allosterically in vitro (Dholakia et al.,
1986; Gross et al., 1988; Gross and Rubino, 1989; Oldfield and
Proud, 1992; Singh et al., 1994; Kimball and Jefferson, 1995).
However, it is not clear whether the concentrations of these compounds
present in vivo (or changes in their concentration following
cell stimulation) are in the range required to modulate eIF-2B
activity. Furthermore, the assays performed in this and the other
studies cited above entailed extensive dilution of the samples relative
to the intracellular milieu, and these low affinity ligands are likely
to be diluted out beyond the level at which they exert their allosteric
effects. Another possible mechanism is the phosphorylation of eIF-2B
itself. It is phosphorylated in vitro by at least three
protein kinases (casein kinases-1 and -2 and glycogen synthase
kinase-3), phosphorylation by each of which may modulate the exchange
activity of eIF-2B (Dholakia and Wahba, 1988; Welsh and Proud, 1993;
Denslow et al., 1994).
Phosphorylation by glycogen
synthase kinase-3 appears to inhibit the activity of eIF-2B, while the
other two are reported to activate. However, it is not clear whether
the activities of the casein kinases are altered following glucose
stimulation of islets and in our hands neither of these kinases has any
measurable affect on eIF-2B activity (Oldfield and Proud, 1992). In the
present work we found that glycogen synthase kinase-3 activity and the
activity of MAP kinase, a potential upstream regulator of glycogen
synthase kinase-3 (Sutherland et al., 1993; Cross et
al., 1994; Sutherland and Cohen, 1994; Welsh et al.,
1994), were not altered under conditions of elevated external glucose.
Thus it is unclear how the changes in the level of eIF-2B activity are
brought about.
Other translation factors are also believed to play
key roles in the control of translation, especially those interacting
with mRNA, which may modulate translation in a selective manner
(Manzella et al., 1991; Rhoads, 1993; Sonenberg, 1993). Two
such factors are eIF-4E and 4E-BP1, which are most likely to control
the translation of mRNAs whose 5`-untranslated regions are rich in
secondary structure. However, no changes in the phosphorylation states
of either protein, or in their association with one another, were
observed in these studies. They do not therefore appear likely to be
important in the regulation of preproinsulin synthesis in response to
glucose. Although the 5`-untranslated region of the preproinsulin mRNA
does contain potential stem loops, these are relatively small
(G = -8 to -13 kcal/mol depending on
species; see Knight and Docherty(1992)) compared to those known to
significantly influence translational efficiency (Kozak, 1989, 1991;
Pelletier and Sonenberg, 1985). Thus is is unlikely that these
potential secondary structure elements play a role in controlling
preproinsulin synthesis, and it is thus unsurprising that eIF-4E and
4E-BP1 seem not to be involved in this process. The lack of change in
the phosphorylation of eEF-2 also eliminates this protein as being
involved in the glucose-induced regulation of preproinsulin
translation.
In conclusion, it seems possible that the enhancement
of eIF-2B activity contributes to the activation of preproinsulin mRNA
translation caused by glucose although other mechanisms may also be
involved. These include the proteins recently identified as binding to
the 5`-untranslated region of this mRNA (Knight and Docherty, 1992). An
important finding of these studies is that glucose activates the
exchange factor eIF-2B independently of changes in eIF-2
phosphorylation.