In this study we used selective inhibitors of glycogen synthase kinase-3 GSK-3 and an allosteric inhibitor of phosphory-lase CP-91149 that causes dephosphorylation of phos-phorylase a, t
Trang 1Inactivation of phosphorylase is a major component of the mechanism
by which insulin stimulates hepatic glycogen synthesis
Susan Aiston1, Matthew P Coghlan2* and Loranne Agius1
1
School of Clinical Medical Sciences, University of Newcastle upon Tyne, The Medical School, Newcastle upon Tyne, UK;
2
Department of Vascular Biology, GlaxoSmithKline, Harlow, Essex, UK
Multiple signalling pathways are involved in the mechanism
by which insulin stimulates hepatic glycogen synthesis In
this study we used selective inhibitors of glycogen synthase
kinase-3 (GSK-3) and an allosteric inhibitor of
phosphory-lase (CP-91149) that causes dephosphorylation of
phos-phorylase a, to determine the relative contributions of
inactivation of GSK-3 and dephosphorylation of
phos-phorylase a as alternative pathways in the stimulation of
glycogen synthesis by insulin in hepatocytes
GSK-3 inhibitors (SB-216763 and Li+) caused a greater
activation of glycogen synthase than insulin (90% vs 40%)
but a smaller stimulation of glycogen synthesis (30% vs
150%) The contribution of GSK-3 inactivation to insulin
stimulation of glycogen synthesis was estimated to be less
than 20% Dephosphorylation of phosphorylase a with
CP-91149 caused activation of glycogen synthase and
translo-cation of the protein from a soluble to a particulate fraction
and mimicked the stimulation of glycogen synthesis by
insulin The stimulation of glycogen synthesis by phos-phorylase inactivation cannot be explained by either inhi-bition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translo-cation of synthase Titrations with the phosphorylase inac-tivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phos-phorylase a We conclude that a signalling pathway invol-ving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis Selective inactivation of phos-phorylase can mimic insulin stimulation of hepatic glycogen synthesis
Keywords: glycogen synthase kinase-3; glycogen synthesis; insulin; liver; phosphorylase
Insulin, glucose and amino acids are the major
physio-logical regulators of hepatic glycogen synthesis [1–3] The
signalling pathways activated by insulin in hepatocytes
[3–7] bear similarities to the mechanisms identified in other
cell types [8,9] Binding of insulin to the receptor causes
phosphorylation of insulin receptor substrates 1 and 2,
recruitment and activation of phosphatidylinositol
3-kinase, resulting in formation of phosphatidylinositol
3,4,5-trisphosphate, which causes recruitment of
phospha-tidylinositol-dependent kinase-1 to the plasma membrane
[8,9] The latter enzyme phosphorylates and activates
protein kinase B, which in turn phosphorylates and
inactivates glycogen synthase kinase-3 (3) As
GSK-3 causes inactivation of glycogen synthase by
phosphory-lation at three sites, inactivation of GSK-3 allows glycogen
synthase to become activated by dephosphorylation
Stimulation of glycogen synthesis by insulin in hepatocytes
is counteracted by inhibitors of phosphatidylinositol 3-kinase [4–6] and is associated with activation of protein kinase B and inactivation of GSK-3 [5–7] However, the contribution of this signalling pathway to the stimulation
of glycogen synthesis by insulin in hepatocytes has not been determined
An alternative mechanism for regulation of glycogen synthase is through changes in the concentration of phosphorylase a, which inhibits glycogen synthase phos-phatase activity [1] by binding to the C-terminus of the glycogen targeting protein [10] This protein was thought to
be present only in liver, but expression in human skeletal muscle has also been reported [11] Phosphorylase kinase catalyses the conversion of inactive phosphorylase b into active phosphorylase a by phosphorylation of a serine residue at the N-terminus [1] Metabolic conditions that decrease the concentration of phosphorylase a through either inhibition of phosphorylase kinase or activation of phosphorylase phosphatase are expected to reverse the inhibition of glycogen synthase phosphatase by phosphory-lase a (Fig 1) Dephosphorylation of phosphoryphosphory-lase a is
a component of the mechanism by which high glucose concentration causes activation of glycogen synthase [1] Binding of glucose to phosphorylase a makes the enzyme
a better substrate for phosphorylase phosphatase, and the decrease in phosphorylase a reverses the inhibition of glycogen synthase phosphatase [1]
Correspondence to L Agius, School of Clinical Medical Sciences,
The Medical School, University of Newcastle upon Tyne,
Newcastle upon Tyne, NE2 4HH, UK.
Fax: + 44 191 2220723, Tel.: + 44 191 2227033,
E-mail: Loranne.Agius@ncl.ac.uk
Abbreviations: GSK-3, glycogen synthase kinase-3; MEM, minimum
essential medium; PTG, protein targeting to glycogen.
*Present address: Cardiovascular & Gastrointestinal Department,
AstraZeneca, Macclesfield, Cheshire SK10 4TG, UK.
(Received 21 March 2003, revised 30 April 2003, accepted 1 May 2003)
Trang 2There is a long-standing debate as to whether inactivation
of phosphorylase is a component of the mechanism by
which insulin activates glycogen synthase in hepatocytes,
because inactivation of phosphorylase by insulin has been
observed in some but not other studies [3,6] In freshly
isolated hepatocyte suspensions, sustained inactivation of
phosphorylase by insulin has generally been observed only
in the presence of glucagon or other counter-regulatory
hormones However, this experimental model shows small
stimulatory effects of insulin on glycogen synthesis because
of the catabolic state of glycogen turnover [3] Short-term
preculture of hepatocytes with dexamethasone allows
recovery from the catabolic state and restores a large
stimulatory effect of insulin on glycogen synthesis similar to
the stimulation that occurs in vivo [3] The contribution of
phosphorylase inactivation to the stimulation of glycogen
synthesis by insulin in this experimental model has not been
tested
Recently, several potent allosteric inhibitors of
phos-phorylase have been identified by high-throughput screens
[12–14] Some of these compounds inhibit glycogenolysis in
hepatocytes by both allosteric inhibition of phosphorylase
and inactivation (conversion of phosphorylase a to b)
similar to glucose [14], whereas others inhibit glycogenolysis
exclusively by allosteric inhibition [15,16] or by inactivation
[16] The latter compounds are very powerful experimental
tools to investigate the role of the phosphorylation state of
phosphorylase in metabolic control [12,17] We demonstrate
in this study, using selective inhibitors of GSK-3 [18–20]
and a selective inhibitor of phosphorylase [12] that causes
dephosphorylation of phosphorylase a [16], that
inactiva-tion of GSK-3 in the absence of phosphorylase inactivainactiva-tion
is a small component of the mechanism by which insulin
stimulates hepatocyte glycogen synthesis In contrast,
dephosphorylation of phosphorylase can mimic insulin action and is a major component of the mechanism by which insulin stimulates glycogen synthesis
Materials and Methods
Materials CP-91149 [12] and SB-216763 [18] were gifts from Pfizer Global Research & Development, Groton Laboratories,
CT, USA, and SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK, respectively The adenovirus vectors for expression of wild-type GSK-3 and S9A-GSK-3 [21] were kindly provided by M Birnbaum, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia,
PA, USA
Hepatocyte isolation and cell culture Hepatocytes were isolated by collagenase perfusion of the liver of male Wistar rats (body weight 180–280 g) obtained from B & K, Hull, UK [22] They were suspended in minimum essential medium (MEM) supplemented with 7% (v/v) newborn calf serum, and plated in multiwell plates After cell attachment ( 4 h), the medium was replaced with serum-free MEM containing 5 mMglucose and 10 nM dexamethasone, and the hepatocytes were cultured for 16–18 h [17]
Treatment of hepatocytes with recombinant adenoviruses
At 2 h after plating, the hepatocytes were incubated for 2 h
in serum-free MEM without or with various titres of either
Fig 1 Model showing two alternative mechanisms for the activation of hepatic glycogen synthase by insulin involving either inactivation of GSK-3
or inactivation of phosphorylase Insulin phosphorylates and inactivates (–) GSK-3 by activation of protein kinase B (PKB) GSK-3 (+, dephosphorylated form) phosphorylates and inactivates glycogen synthase (GS) Dephosphorylation (activation) of glycogen synthase (GS-b)
by synthase phosphatase (SP) is inhibited by phosphorylase a (Phos-a) Conversion of phosphorylase a into phosphorylase b (Phos-b) by phosphorylase phosphatase (PP) is stimulated by glucose and by CP-91149 Insulin may convert phosphorylase a into phosphorylase b by either inhibition of phosphorylase kinase (PK) or activation of phosphorylase phosphatase This reverses the inhibition of synthase phosphatase
by phosphorylase a.
Trang 3AdCMV-GSK-3 or AdCMV-S9A-GSK-3 for expression of
wild-type or mutant GSK-3, respectively [21] The medium
was then replaced with serum-free MEM containing
dexamethasone, and the cells were cultured for 16–18 h
as above Overexpression of GSK-3 was confirmed by
immunoblotting after 18 h of culture
Incubations with insulin and inhibitors
After preculture for 16–18 h in MEM with
dexametha-sone, the medium was replaced with fresh MEM without
dexamethasone and with the substrates and inhibitors
indicated Parallel incubations were performed for
deter-mination of glycogen synthesis, glycogen synthase and
phosphorylase a activity For determination of glycogen
synthesis, hepatocyte monolayers were incubated for 3 h
in MEM containing [U-14C]glucose (2 lCiÆmL)1) and the
glucose concentrations indicated Inhibitors were dissolved
in dimethylsulfoxide, and control incubations contained
an equivalent volume (0.1%, v/v) of dimethylsulfoxide
Radiolabelled glycogen was determined as described
previously [22] Glycogen synthesis is expressed as nmol
of glucose incorporated into glycogenÆ3 h)1Æ(mg cell
pro-tein))1
Enzyme assays and immunoblotting
At the end of the incubation, the plates were
snap-frozen in liquid nitrogen and stored at )80 °C until
assay For determination of glycogen synthase, cells were
extracted as previously described [23], and assays were
performed on the whole homogenate (unless indicated
otherwise) or in the 13 000 g supernatant and pellet
fractions from the incorporation of UDP[6-3H]glucose
into glycogen in the absence or presence of 6.7 mM
Glc6P, representing active and total glycogen synthase,
respectively [24] Active glycogen synthase (– Glc6P) is
expressed either as mUÆ(mg protein))1 (nmolÆmin)1Æmg)1)
or as the activity ratio (– Glc6P/+ Glc6P) For
deter-mination of phosphorylase a, the hepatocytes were
extracted as described previously [17] Phosphorylase a
was determined in the supernatant spectrometrically
by coupling to phosphoglucomutase and
glucose-6-phosphate dehydrogenase [23] Phosphorylase a activity
is expressed as mUÆ(mg cell protein))1 (nmolÆ
min)1Æmg)1) GSK-3 was assayed as in [5] Activity is
expressed as pmol32P incorporatedÆmin)1Æ(mg protein))1
Immunoblotting for glycogen synthase and GSK-3 was
performed after fractionation of the extracts by SDS/
PAGE After transfer of the proteins to nitrocellulose,
the membrane was probed with a rabbit antibody to rat
liver glycogen synthase raised against residues IP
KGKKKLHGEYKN(690–703) [25] or a goat antibody
to GSK-3b (Santa Cruz, Santa Cruz, CA, USA)
followed by incubation with the appropriate
peroxi-dase-conjugated secondary antibody (Jackson
Immuno-research, West Grove, PA, USA) Immunoreactive
bands were visualized using an ECL kit (Amersham
Biotech)
Results are expressed as means ± SEM for the number
of hepatocyte preparations indicated Statistical analysis
was by Student’s paired t test
Results
Insulin causes rapid and sustained inactivation
of phosphorylase When hepatocytes were precultured as described in Mate-rials and Methods and then incubated in fresh MEM without dexamethasone, insulin caused a rapid and sus-tained decrease in the activity of phosphorylase a at both
5 mM glucose (40% decrease) and 25 mM glucose (60% decrease) The inactivation by insulin was observed within
15 min and had a similar time course at low and high glucose (Fig 2A) This contrasts with the activation of
Fig 2 Time course of inactivation of phosphorylase a (A) and activa-tion of glycogen synthase (B) by 10 n M insulin Hepatocytes were incubated with the glucose concentrations indicated for 4 h and with
10 n M insulin for the time intervals indicated Values are means ± SEM, n ¼ 4–6 *P < 0.05; **P < 0.005 inactivation by insulin relative to control.
Trang 4glycogen synthase by insulin, which was more rapid at
25 mMthan at 5 mMglucose (Fig 2B)
Inhibition of GSK-3 is less effective than insulin
at stimulating glycogen synthesis
Inactivation of GSK-3 by insulin in hepatocytes has been
demonstrated previously [5,6] In this study we confirmed
that insulin inactivates GSK-3 (control, 0.21 ± 0.04;
10 nM insulin for 10 min, 0.14 ± 0.04, n¼ 10, pmolÆ
min)1Æmg)1, P < 0.05) The inactivation by insulin (33%)
was less than the inactivation caused by calyculin A
(50 nM), a protein phosphatase inhibitor (0.025 ± 0.01,
88%, P < 0.05), indicating that insulin causes only partial
inactivation of GSK-3 To determine the role of GSK-3
inactivation in insulin action, we used the selective GSK-3
inhibitor 216763, an arylindolemaleimide [18]
SB-216763 caused a concentration-dependent increase in the
activity ratio of glycogen synthase (Fig 3A), in agreement
with previous findings on other cell types [18], and it had
no significant effect on the activity of phosphorylase a
(control, 7.0 ± 2.7; 10 lM SB-216763, 6.0 ± 2.1; 25 lM
SB-216763, 5.8 ± 1.9, n¼ 4, mUÆmg)1) When compared
with insulin, SB-216763 caused a larger activation of
glycogen synthase (93% vs 40%, Fig 3B) but a smaller
stimulation of glycogen synthesis (28% vs 156%,
Fig 3C) Similarly 10 mM Li+, a potent inhibitor of
GSK-3 [19,20], also caused a larger activation of glycogen
synthase (73% vs 40%) but a smaller stimulation of
glycogen synthesis (28% vs 156%) than insulin In the
combined presence of insulin and either SB-216763 or Li+, the activation of glycogen synthase was greater (P < 0.05) than with GSK-3 inhibitors alone, indicating that insulin activates glycogen synthase by mechanisms additional to inactivation of GSK-3 (Fig 3B), and it was significantly (P < 0.05) greater than with insulin alone, consistent with the partial inactivation of GSK-3 by insulin The rates of glycogen synthesis in the combined presence of GSK-3 inhibitors and insulin were the same as with insulin alone, despite the further activation of glycogen synthase (Fig 3C)
Inactivation of phosphorylase mimics insulin stimulation of glycogen synthesis
We used CP-91149, a potent selective inhibitor of phos-phorylase [12] that causes conversion of phosphos-phorylase a to
bin hepatocytes [16,17], to determine the role of dephos-phorylation of phosphorylase in the regulation of glycogen synthesis CP-91149 (2.5 lM) caused a similar stimulation of glycogen synthesis and inactivation of phosphorylase to that
of insulin (Fig 4) It is noteworthy that 1,4-dideoxy-1,4-imino-D-arabinitol, an allosteric inhibitor of phosphorylase [26] that does not cause dephosphorylation of phosphory-lase a [16], does not stimulate glycogen synthesis [15] This was confirmed in parallel incubations in the present study (results not shown), indicating that stimulation of glycogen synthesis by CP-91149 is due to dephosphorylation of phosphorylase a rather than inhibition of glycogen degra-dation or cycling
Fig 3 Effects of insulin and GSK-3 inhibitors on glycogen synthase and glycogen synthesis Hepatocytes were incubated for 3 h in MEM containing
10 m M glucose and the additions indicated (A) Activation of glycogen synthase by various concentrations of SB-216763 (B) and (C) Effects of
25 l M SB-216763 or 10 m M LiCl in the absence (open bars) or presence (closed bars) of 10 n M insulin on the activity of glycogen synthase and rates
of glycogen synthesis Incubation mixtures for determination of glycogen synthesis contained [U- 14 C]glucose as described in Materials and methods The total activity of glycogen synthase assayed in the presence of Glc6P was 1.05 ± 0.16 (A) and 0.94 ± 0.14 (B) mUÆmg)1and was not affected by the incubation conditions tested Values are means ± SEM, n ¼ 4 *P < 0.05 relative to no insulin; #P < 0.05 relative to no inhibitor.
Trang 5Stimulation of glycogen synthesis but not activation
of glycogen synthase by insulin can be explained
by inactivation of phosphorylase
To determine the role of inactivation of phosphorylase in
the stimulation of glycogen synthesis by insulin, we
determined the combined effects of insulin and various
concentrations of CP-91149 The effects of insulin on
phosphorylase inactivation, glycogen synthase activation
(Fig 5A), and stimulation of glycogen synthesis (Fig 5B)
were significant at all concentrations of inhibitor tested
(P < 0.05) When the activity of glycogen synthase was
plotted against the respective activity of phosphorylase a,
the curves for untreated (control) and insulin-treated
incubations were not superimposed (Fig 5C), indicating
that activation of glycogen synthase by insulin cannot be
fully explained by inactivation of phosphorylase However,
the corresponding curves for glycogen synthesis against
phosphorylase a (Fig 5D) in the absence and presence of
insulin were superimposed over a wide range of activities of
phosphorylase down to 40% of basal activity This indicates that insulin stimulates glycogen synthesis predominantly by inactivation of phosphorylase over the range 3–7 mUÆmg)1 but also by additional mechanisms at activities below
3 mUÆmg)1
The stimulation of glycogen synthesis by insulin and phosphorylase inactivation are greater than can be explained by activation of glycogen synthase
In view of the above findings that inactivation of phosphorylase mimics insulin stimulation of glycogen synthesis (Figs 4 and 5), whereas inactivation of GSK-3 has only a small stimulatory effect despite the large activation
of glycogen synthase (Fig 3), we tested the relative roles of GSK-3 and phosphorylase a in the same experiments by modulating GSK-3 activity by adenovirus-mediated expres-sion of wild-type GSK-3 or a constitutively active mutant (S9A-GSK-3) [21,27] or by inhibiting GSK-3 activity with SB-216763 (Fig 6) Incubations for determination of glyco-gen synthesis were performed in the absence or presence of insulin or CP-91149, and rates of glycogen synthesis were expressed relative to the corresponding activity of glycogen synthase (assayed in the absence of Glc6P)
In the absence of insulin or CP-91149, the relation between the rate of glycogen synthesis and the activity of glycogen synthase was sigmoidal (Fig 6, solid line) In cells expressing endogenous GSK-3, the rate of glycogen synthesis was at or near the plateau of the sigmoidal curve The GSK-3 inhibitor caused a twofold increase in the activity of glycogen synthase, but had little effect on flux Conversely, GSK-3 overexpression caused a marked decrease in both the activity of glycogen synthase and the rate of glycogen synthesis Both insulin (dashed line) and CP-91149 (dotted line) caused an upward shift in the glycogen synthesis versus glycogen synthase curve, indica-ting that stimulation of glycogen synthesis cannot be explained by activation of glycogen synthase alone
Inactivation of phosphorylase causes translocation
of glycogen synthase
To test for other mechanisms that might explain the stimulation of glycogen synthesis by phosphorylase inacti-vation, we determined the effects of CP-91149 on the subcellular distribution of glycogen synthase Previous studies showed that high glucose concentration causes translocation of glycogen synthase from a soluble to a particulate fraction by a Glc6P-dependent mechanism [28]
We therefore determined the distribution of glycogen synthase total activity (assayed in the presence of Glc6P)
or protein (by immunoblotting), between the supernatant and particulate fractions in cells treated with CP-91149 or various concentrations of glucose and insulin CP-91149 caused translocation of glycogen synthase from the super-natant to the particulate fraction, as shown by immuno-blotting (Fig 7A) or the radiochemical assay (Fig 7B), similar to the combined effect of 25 mMglucose and insulin (Fig 7B) As phosphorylase a and glycogen synthase bind
to the glycogen targeting protein (PTG) by a mutually exclusive mechanism [29,30], we tested for the presence of PTG in the particulate fraction Immunoreactivity to PTG
Fig 4 Effects of insulin and CP-91149 on phosphorylase a (A) activity
and glycogen synthesis (B) at various glucose concentrations
Hepato-cytes were incubated for 3 h with the glucose concentrations indicated
in the absence (s) or presence of 10 n M insulin (j) or 2.5 l M
CP-91149 (m) Values are means ± SEM, n ¼ 4 Effects of insulin and
CP-91149 were significant (P < 0.05) at all concentrations of glucose.
Trang 6(at 36 kDa) was detected in both supernatant and the
particulate fractions (results not shown)
Discussion
Inactivation of GSK-3 is considered to be a key component
of the mechanism by which insulin stimulates glycogen
synthesis [8,9] However, the quantitative contribution of
this mechanism compared with other signalling pathways to
the stimulation of glycogen synthesis by insulin has not
been evaluated In this study we used selective inhibitors of
GSK-3 [18–20] and a selective inhibitor of phosphorylase
that causes dephosphorylation of the enzyme [12,16] to
determine the contributions of inactivation of GSK-3 and
dephosphorylation of phosphorylase to the mechanism by
which insulin stimulates glycogen synthesis in liver cells
Three key conclusions can be drawn: (a) that inactivation of
phosphorylase is an essential component of the mechanism
by which insulin stimulates glycogen synthesis and it can
account for the stimulation of glycogen synthesis by insulin
over a wide range of activities of phosphorylase a; (b) that
suppression of GSK-3 activity, in the absence of
inactiva-tion of phosphorylase, causes a large activainactiva-tion of glycogen synthase but a small stimulation of glycogen synthesis; (c) that the stimulation of glycogen synthesis by inactivation of phosphorylase is associated with both activation and translocation of glycogen synthase, and that the former mechanism alone cannot explain the stimulation of glyco-gen synthesis This suggests that translocation of glycoglyco-gen synthase may be an essential component of the mechanism
by which dephosphorylation of phosphorylase leads to stimulation of glycogen synthesis
GSK-3 inactivation has been implicated in the mechan-ism by which insulin stimulates glycogen synthesis on the basis of three pieces of evidence First, inactivation of GSK-3
by insulin occurs in several cell types [8,9] Second, GSK-3 causes phosphorylation and inactivation of glycogen syn-thase whereas inhibition of GSK-3 in intact cells causes activation of glycogen synthase [21] Third, overexpression
of a constitutively active GSK-3 mutant overrides the activation of glycogen synthase [27] and stimulation of glycogen synthesis caused by insulin in adipocytes [21] In the present study a clear role for GSK-3 in the regulation of glycogen synthase has been demonstrated by overexpression
Fig 5 Stimulation of glycogen synthesis but not activation of glycogen synthase by insulin can be largely explained by inactivation of phosphorylase Hepatocytes were incubated for 3 h with 15 m M glucose and the concentrations of CP-91149 indicated in either the absence (open symbols) or presence (closed symbols) of 10 n M insulin (A) Phosphorylase a and active glycogen synthase (– Glc6P) expressed as mUÆ(mg protein))1 (B) Glycogen synthesis (C) Active glycogen synthase vs phosphorylase a (D) Glycogen synthesis vs phosphorylase a Values are means ± SEM,
n ¼ 4.
Trang 7of wild-type or S9A-GSK-3, which caused marked
inacti-vation of glycogen synthase, and by the GSK-3 inhibitors,
which caused twofold activation of glycogen synthase The
greater activation of glycogen synthase by GSK3 inhibitors
(90%) compared with insulin (40%) can be explained by the
small fractional inactivation of GSK-3 caused by insulin
(33%) Whereas inactivation of glycogen synthase by GSK-3
overexpression resulted in inhibition of glycogen synthesis,
activation of glycogen synthase by GSK-3 inhibition had
little effect on glycogen synthesis This is explained by
the sigmoidal relation between glycogen synthesis and
glycogen synthase activity at endogenous activities of
phosphorylase a Thus GSK-3 inactivation in the absence
of inactivation of phosphorylase had little impact on flux
through glycogen synthesis in hepatocytes, despite
activa-tion of glycogen synthase Accordingly the inactivaactiva-tion of
GSK-3 caused by insulin accounted for less than 20% of the
stimulation of glycogen synthesis The additive activation of
glycogen synthase by insulin and GSK-3 inhibitors indicates
that insulin activates glycogen synthase by mechanisms
additional to inactivation of GSK-3 This can be explained,
at least in part, by the inactivation of phosphorylase by
insulin
Two pieces of evidence from the studies with the
phosphorylase inhibitor (CP-91149) support a role for
dephosphorylation of phosphorylase a as a critical compo-nent of the mechanism by which insulin stimulates glycogen synthesis In the absence of insulin, the phosphorylase inhibitor CP-91149 caused a dose-dependent stimulation of glycogen synthesis that exceeded the stimulation caused by insulin, at concentrations of inhibitor that inactivated phosphorylase a by 85% This stimulation of glycogen synthesis cannot be explained by inhibition of glycogen degradation or by a decrease in catalytic activity of phosphorylase, because it is not mimicked by another selective inhibitor of phosphorylase that inhibits glucagon-stimulated glycogenolysis [15,16] but does not cause dephosphorylation of phosphorylase a [16] This lack of stimulation of glycogen synthesis by an allosteric inhibitor
of phosphorylase [15,31] is further evidence against cycling between glycogen synthesis and degradation as shown also
in other in vitro models [32] The stimulation of glycogen synthesis by CP-91149 also cannot be explained by nonspecific effects on glucose metabolism because the
Fig 6 Relation between glycogen synthesis and active glycogen
syn-thase determined by modulation of GSK- 3 activity by GSK- 3
overex-pression or inhibition (A) GSK-3b was determined by immunoblotting
of hepatocytes that were either untreated (End) or treated with
wild-type AdCMV-GSK-3 (w) or mutant AdCMV-S9A-GSK-3 (m) and
cultured for 18 h (B) Hepatocytes were either untreated (open
sym-bols) or treated (closed symsym-bols) with AdCMV-GSK-3 (w) or
AdC-MV-S9A-GSK-3 (m) After 18 h of culture, they were incubated for
3 h in medium containing 10 m M glucose without (s,d) or with 10 n M
insulin (h,j) or 2.5 l M CP-91149 (n,m) for determination of
glyco-gen synthesis and active glycoglyco-gen synthase (– Glc6P) Where indicated
(Inh), 25 l M SB-216763 was added during the 3 h incubation to inhibit
GSK-3 activity Glycogen synthesis is plotted against the respective
glycogen synthase activity Values are means ± SEM, n ¼ 4.
Fig 7 Inactivation of phosphorylase causes translocation of glycogen synthase Hepatocytes were incubated for 60 min with 5 m M glucose (A) or the glucose concentrations indicated (B) without (Con) or with
10 l M CP-91149 (CP) or 10 n M insulin (Ins) The cell homogenates were centrifuged at 13 000 g, and total glycogen synthase (GS) was determined in the supernatant (SN) and pellet (P) fractions by immunoblotting (A) or radiochemically (B) (A) Immunoblot and corresponding densitometry (B) Total glycogen synthase activity (assayed in the presence of Glc6P) in the pellet as a percentage of supernatant plus pellet activity Values are means ± SEM, n ¼ 3.
Trang 8compound had no effect on glucose phosphorylation,
glycolysis, or the Glc6P content of hepatocytes [17] The
stimulation of glycogen synthesis by CP-91149 therefore
shows that inactivation (dephosphorylation) of
phosphory-lase can mimic the stimulatory effect of insulin
In the experiments in which the effects of insulin on
glycogen synthase and synthesis were tested in the presence
of various concentrations of the phosphorylase inhibitor
and expressed relative to the respective activity of
phos-phorylase a, the stimulation of glycogen synthesis but not
the activation of glycogen synthase could be largely
accounted for by inactivation of phosphorylase over a wide
range of activities of phosphorylase a (> 3 mUÆmg)1)
These findings are in agreement with the high flux control
coefficient of phosphorylase on glycogen synthesis [17] and
with the GSK-3 inhibitor studies that show a sigmoidal
relation between glycogen synthesis and glycogen synthase
activity, with a basal rate of flux at or near the plateau
The phosphorylase inactivator, CP-91149, caused both
activation of glycogen synthase and translocation of the
enzyme to the pellet Previous studies have shown that high
glucose concentration causes translocation of glycogen
synthase in hepatocytes to the cell periphery [25] and from
the supernatant to the particulate fraction [28] This effect of
glucose is abolished by inhibition of glucose
phosphoryla-tion and correlates with the accumulaphosphoryla-tion of Glc6P [28],
suggesting a role for Glc6P in translocation of glycogen
synthase However, in some metabolic conditions,
translo-cation of glycogen synthase does not correlate with Glc6P
[33] Two explanations are possible Either there is
subcel-lular compartmentation of Glc6P [34] or additional
mech-anisms may be involved in mediating translocation of
glycogen synthase CP-91149 does not affect the Glc6P
content of hepatocytes [17] Although we cannot rule out
subcellular changes in the concentration of Glc6P in the
presence of the phosphorylase inactivator, we suggest that
additional factors may be involved in translocation of
glycogen synthase and that phosphorylase a itself may be an
important determinant of the subcellular compartmentation
of glycogen synthase through competitive binding to a
common targeting protein PTG [29] has a binding site at
the C-terminus, which binds phosphorylase a and glycogen
synthase by a mutually exclusive mechanism [30] If the
binding affinity of PTG is greater for phosphorylase a than
for phosphorylase b, then dephosphorylation of
phosphory-lase a may favour binding of glycogen synthase
Overex-pression of PTG in hepatocytes activates glycogen synthase
and stimulates glycogen synthesis [35,36] As the twofold
activation of glycogen synthase by inhibition of GSK-3 has a
negligible effect on glycogen synthesis (this study), the
stimulation of glycogen synthesis by PTG overexpression
could be explained by either combined activation of glycogen
synthase and inactivation of phosphorylase [35] or combined
activation of glycogen synthase and translocation or binding
to PTG The hypothesis that inactivation of phosphorylase
by CP-91149 or insulin may cause binding of glycogen
synthase to glycogen targeting proteins remains to be tested
Until now, studies on insulin signalling in relation to
glycogen synthesis have largely focused on mechanisms
leading to activation of protein kinase B and inactivation of
GSK-3 We demonstrate in this study that inactivation of
phosphorylase but not inhibition of GSK-3 mimics insulin
stimulation of glycogen synthesis in hepatocytes and that insulin action on glycogen synthesis can be largely accoun-ted for by phosphorylase inactivation Accordingly, studies
on insulin signalling should address the mechanism that leads to dephosphorylation of phosphorylase In hepatocyte suspensions incubated in the absence of amino acids, insulin activates protein kinase B but does not activate glycogen synthase, suggesting that activation of protein kinase B alone is not sufficient to elicit the anabolic effects of insulin [37] On the basis of the present findings that inactivation of phosphorylase is essential for stimulation of glycogen synthesis by insulin and it is also a contributing factor to the activation of glycogen synthase, the question could be raised whether medium amino acids are either essential for,
or have a permissive role in mediating, the inactivation of phosphorylase by insulin?
Acknowledgements
This work was supported by Diabetes UK and by the Medical Research Council We thank Dr Morris Birnbaum for the gift of GSK-3 adenoviruses, Drs Joan Guinovart and Rez Halse for the antibodies to glycogen synthase a and PTG, respectively, and Dr Judith Treadway for CP-91149 and helpful advice.
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