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Veterans Affairs San Diego Healthcare System, San Diego, California 92161; and Department of Medicine, University of California, San Diego, La Jolla, California 92093
Address all correspondence and requests for reprints to: R. R. Henry, M.D., Veterans Affairs San Diego Healthcare System (9111G), 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: rrhenry{at}vapop.ucsd.edu
Abstract
Insulin signaling pathways potentially involved in regulation of
skeletal muscle glycogen synthase were compared in
differentiated human muscle cell cultures from nondiabetic and type 2
diabetic patients. Insulin stimulation of glycogen synthase activity as
well as phosphorylation of MAPK, p70 S6 kinase, and protein kinase B
(Akt) were blocked by the phosphatidylinositol 3-kinase inhibitors
wortmannin (50 nM) and LY294002 (10 µM). In
contrast to lean and obese nondiabetic subjects, where there were
minimal effects (15–20% inhibition), insulin stimulation of glycogen
synthase in muscle cultures from diabetic subjects was greatly
diminished (
75%) by low concentrations of wortmannin (25
nM) or LY294002 (2 µM). This increased
sensitivity of diabetic muscle to impairment of insulin-stimulated
glycogen synthase activity occurs together with diminished
insulin-stimulation (by 40%) of IRS-1-associated phosphatidylinositol
3-kinase activity in the same cells. Protein expression of IRS-1, p85,
p110, Akt, p70 S6 kinase, and MAPK were normal in diabetic cells, as
was insulin-stimulated phosphorylation of Akt, p70 S6 kinase, and MAPK.
These studies indicate that, despite prolonged growth and
differentiation of diabetic muscle under normal metabolic culture
conditions, defects of insulin-stimulated phosphatidylinositol 3-kinase
and glycogen synthase activity in diabetic muscle persist, consistent
with intrinsic (rather than acquired) defects of insulin action.
SKELETAL MUSCLE GLYCOGEN synthesis plays an important role in blood glucose homeostasis (1, 2, 3). The ability of insulin to increase glucose utilization into muscle involves stimulation of glucose transport and activation of glycogen synthase (GS), the rate-limiting enzyme for glycogen synthesis (4, 5). Both the basal activity of GS and its responsiveness to insulin are impaired in skeletal muscle of type 2 diabetic (T2D) subjects (6, 7, 8, 9) and may play a major role in the development of glucose intolerance and insulin resistance.
Of the multitude of signaling events initiated after insulin binding to its receptor, three candidate pathways have been proposed to play an important role in activating GS. These include: the ras/MAPK/p90rsk, phosphatidylinositol 3kinase (PI 3-kinase)/protein kinase B (Akt), and PI 3-kinase/mTOR/p70 S6 kinase pathways (10, 11, 12, 13, 14, 15, 16, 17). In several cell lines, structurally unrelated inhibitors of PI 3-kinase (wortmannin and LY294002) inhibit insulin stimulation of GS (18, 19, 20, 21, 22), suggesting that PI 3-kinase-dependent pathways might mediate this insulin response. Events downstream of PI 3-kinase and the relative importance of these pathways in mediating insulin stimulation of GS seem to be both tissue- and species-dependent. For example, constitutively active Akt has been shown to stimulate glycogen synthesis and elevate GS activity in L6 myotubes but not in 3T3L-1 adipocytes (23). Inhibition of p70 S6 kinase activation blunts insulin stimulation of GS in 3T3-L1 adipocytes (21) and rat skeletal muscle (24) but is ineffective in rat adipocytes (25), PC12 cells (18), skeletal muscle in mice (26), and human myotubes (12). Furthermore, in skeletal muscle of GTG-obese mice, p70 S6 kinase seems to participate in insulin regulation of GS, contrary to the situation in normal mice (26). In light of this tissue and species specificity, it is critical to study impaired insulin signaling and insulin resistance of diabetes in human skeletal muscle.
There are several lines of evidence that implicate inherited factors in the impairments of regulation of glycogen synthesis in type 2 diabetes. Abnormal activation of glycogen synthesis by insulin is retained in cultured fibroblasts (27) and skeletal muscle cells from T2D patients (9, 28). The nature of this impairment is still unknown and may involve defects in signaling pathways leading to activation of GS. In support of this conjecture, studies of muscle biopsy samples after in vivo insulin stimulation showed decreased activation of IRS-1-associated PI 3-kinase activity in muscle of T2D subjects, in comparison with control nondiabetic subjects (29, 30, 31).
As we reported earlier, human skeletal muscle cells, when fully differentiated, display morphological, biochemical, and metabolic properties characteristic of adult skeletal muscle (9, 32). Cells from T2D subjects demonstrate impaired GS activity, in both the absence and presence of insulin (9), that is highly reflective of defects seen in muscle biopsies (33) and thus provides a powerful tool for the investigation of potential intrinsic defects associated with type 2 diabetes, independent of the influence of chronic hyperglycemia, hyperinsulinemia, and other metabolic abnormalities.
To test the hypothesis that the defects in GS activity observed in muscle cultures from T2D subjects could be the result of impaired activation of intermediate steps involved in insulin signal transduction pathways, we compared the effect of inhibitors of the p70 S6 kinase, MAPK, and PI 3kinase pathways on GS activity. In addition, we investigated protein expression and insulin responses of these enzymes. These studies demonstrate that the major portion of insulin’s effect on GS occurs through activation of PI 3-kinase. This activation is impaired in muscle cells from T2D subjects. Persistence of these defects in diabetic muscle cultures under eumetabolic conditions is consistent with intrinsic defects of insulin action.
Materials and Methods
Materials
Glycogen, wortmannin, rapamycin, pepstatin, leupeptin, phenylmethylsulfonylfluoride, and other reagents and chemicals were purchased from Sigma (St. Louis, MO). LY 294002 was from Calbiochem (San Diego, CA). PD098059 was a kind gift from Dr. A. Saltiel, Parke-Davis Pharmaceuticals (Ann Arbor, MI). Antibodies against phospho-p44/42 MAPK and phospho-Akt were obtained from New England Biolabs, Inc. (Beverly, MA). Affinity-purified rabbit polyclonal IRS-1, p85, and p70 S6 kinase antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and the anti-Akt and anti-p110 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sources of other common reagents have been detailed in previous publications (9, 33).
Subjects, human muscle cell culture, and cell treatment
Muscle biopsies for skeletal muscle cultures were obtained from
11 healthy lean, 7 obese nondiabetic (OND), and 11 T2D patients. All
subjects underwent a 2-h 75-g oral glucose tolerance test. Normal
glucose tolerance was defined as a fasting plasma glucose level less
than 110 mg/dl and a 2-h glucose level less than 140 mg/dl
(34). Insulin action or resistance was determined in each
subject, using a 3-h maximal insulin-stimulated hyperinsulinemic (300
mU/m2·min) euglycemic (5.0 mM)
glucose clamp. Glucose disposal rates (GDR) were measured during the
last 30 min of the clamp procedure. The experimental protocol was
approved by the Committee on Human Investigation of the University of
California, San Diego. Informed written consent was obtained from all
subjects after explanation of the protocol. Subject characteristics are
summarized in Table 1
.
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20 pM) for 4–6 wk from the
time of biopsy to actual analysis. All studies were carried out on
first-passage differentiated cultures. On the day of assay,
differentiated cells were placed in serum-free 
-MEM media for 1
h before further incubation. Cells were then incubated with varying
concentrations of wortmannin (25 or 50 nM), LY294002 (2 or
10 µM), rapamycin (50 nM), or PD098059 (50
µM) for 30 min, followed by a 30-min (if not otherwise
indicated) incubation with 30 nM insulin. Previous studies
(9, 28) have established this dose of insulin as that that
provides maximal stimulation of metabolic activities in both
nondiabetic and diabetic muscle cells. Though human muscle cells also
contain IGF-1 receptors, this insulin concentration displaces
approximately 1% of specifically bound
125I-IGF-1 (28), making it unlikely
that insulin action is mediated by the IGF-1 receptor. Wortmannin is a
potent, cell-permeable, and irreversible inhibitor of PI 3-kinase
with IC50 
5 nM in purified
preparations and cell cultures that, at concentrations lower than 100
nM, is selective for PI 3-kinase. LY 294002 is a
cell-permeable, potent and specific PI 3-kinase inhibitor
(IC50 1.4 µM) that acts on
the ATP-binding site of the enzyme. PD098059 is a selective and
cell-permeable inhibitor of MAP kinase kinase that acts by inhibiting
the activation of MAP kinase and subsequent phosphorylation of MAP
kinase substrates. Rapamycin is an antifungal immunosuppressant that,
acting on mTOR, selectively inhibits the phosphorylation and activation
of p70S6 kinase (IC50 = 50 pM)
(35). GS activity
The activity of GS in response to hormone and/or drug treatment was measured as described in detail previously (9). Differentiated human muscle cells were incubated with inhibitory agents and insulin in media containing 5 mM glucose and solubilized as described previously (36). GS activity was assayed at a physiologic substrate concentration (0.3 mM UDP-[14C] glucose) in parallel incubations with 0.1 or 10 mM glucose-6-phosphate. GS activity is expressed as nmol UDP-glucose incorporated into glycogen/min-1.mg total protein or as fractional velocity (FV; the ratio of activity at 0.1 mM G-6-P/10 mM G-6-P). Previous studies have indicated that the FV, as measured in this assay, is an indicator of the activation state of the enzyme (33, 36). Total cellular protein was determined by the Bradford method (37), with BSA as a standard.
Determination of IRS-1-associated PI 3-kinase activity
The assay performed was a modification of that described by Kelly and Ruderman (38). Cells were lysed in buffer containing 20 mM Tris-HCl, 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% NP-40, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 200 µM phenylmethylsulfonylfluoride, 1 µM leupeptin, 1 µM pepstatin, 10 mg/ml aprotinin, pH 7.5. Overnight incubation of lysates (equal amounts of protein) with IRS-1 antibody was followed by 2 h of incubation with protein A-agarose; all steps were performed at 4 C. The immunoprecipitates were washed, and PI 3-kinase activity was measured, as described (39), and quantitated by scanning densitometry. Preliminary studies found that only a small portion of insulin-stimulated PI 3-kinase activity was associated with IRS-2 (<10% of that associated with IRS-1), so attention was focused on the component associated with IRS-1.
Measurements of p85, p110, and IRS-1 protein levels
Protein levels of IRS-1, and the p85 and p110 subunits of PI 3-kinase were measured in the same cell lysates as were used for determination of PI 3-kinase activity. Western blot analysis was performed by the method of Burnette (39). After separation of proteins by SDS/PAGE (10%), proteins were transferred to nitrocelluose membranes and blocked to reduce nonspecific binding with TBS containing 5% nonfat dry milk and 0.05% Tween 20. In all cases, the secondary antibody was antirabbit IgG conjugated with horseradish peroxidase. Proteins were visualized with SuperSignal-enhanced chemiluminescence reagent (Pierce Chemical Co., Rockford, IL) and exposed to autoradiograph film (Hyperfilm, Amersham Pharmacia Biotech, Arlington Heights, IL). The intensity of the specific protein bands was quantified by densitometry (NIH Image).
Determination of MAPK, p70 S6 kinase, and Akt phosphorylation
Changes in the phosphorylation state of MAPK, in response to insulin and drug treatments, were detected using phospho-specific antibodies that selectively interact with two different phosphorylated forms of MAPK: extracellular regulated kinase (ERK)-1 (44 kDa) and ERK-2 (42 kDa) (New England Biolabs, Inc.). Phosphorylation of Akt as a marker of enzyme activation was detected using an antibody raised against a phosphopeptide (Ser473) sequence conserved in Akt (1/2/3) isoforms (New England Biolabs, Inc.). The activation state of p70 S6 kinase was evaluated using antibodies specific to phospho-Thr389 and Ser411-Thr421 (New England Biolabs, Inc.). Lysates from human skeletal muscle cells were analyzed by Western blot after separation of proteins by SDS/PAGE (8–10%). The secondary antibody was antirabbit IgG conjugated with horseradish peroxidase. Proteins were visualized with enhanced chemiluminescence and exposed to autoradiograph film. The intensity of the bands was quantified by densitometry. After blotting with phospho-specific antibodies, membranes were stripped and reprobed with antibodies against the total protein, to ensure equal loading and transfer of protein.
Calculations and statistical methods
All data and calculated results are expressed as means ± SEM. All calculations on cells were performed using paired controls. Paired t test analysis was performed for comparisons of exposures to different inhibitors and/or insulin in the same sets of cells. Significance was accepted at the P < 0.05 level. Because of limitations in tissue availability, not all studies were performed on cells from all subjects. The number of individuals involved in each experiment is indicated in the figure legends.
Results
In vivo and in vitro insulin action in experimental subjects
Subjects who provided muscle tissue for culture were matched for
age. Obese nondiabetic and T2D subjects were also matched for body mass
index. Diabetic subjects displayed elevated fasting glucose, insulin,
and glycosylated hemoglobin A1c levels (Table 1
). Insulin
resistance in that group was confirmed by lower GDR, compared with both
nondiabetic groups, during a 3-h hyperinsulinemic euglycemic clamp
(Table 1). Obese nondiabetic subjects displayed moderate insulin
resistance, indicated by intermediate GDR in response to insulin,
compared with lean nondiabetic (LND) and T2D subjects (Table 1).
The activity of GS in muscle cells, expressed as FV, behaved much like
whole-body glucose disposal. Both basal and insulin-stimulated GS FV in
cells of diabetic subjects were significantly impaired, compared with
the LND group (basal: 0.059 ± 0.008 vs. 0.103 ±
0.008, P < 0.05; insulin-stimulated: 0.109 ±
0.019 vs. 0.183 ± 0.019, P < 0.05),
in agreement with previously published results (9). The
insulin-stimulated increment in FV (Fig. 1), calculated as the difference in GS FV
measured in basal and insulin-stimulated conditions, was reduced by
approximately 30–40% in cells of diabetic patients (0.084 ±
0.01 vs. 0.049 ± 0.01, nondiabetic and T2D,
respectively; P < 0.05). In OND cells, both basal and
insulin-stimulated GS FV were intermediate between LND and diabetic
groups (basal: 0.087 ± 0.020; insulin-stimulated: 0.137 ±
0.021), as well as the insulin-stimulated increment in GS FV
(0.061 ± 0.015).
|
Treatment of cells, from nondiabetic and diabetic patients, with
50 nM wortmannin did not affect basal total activity of GS
(measured at a saturating concentration of the allosteric activator
glucose-6-phosphate) in either group of subjects (2.63 ± 0.37
nmol/mg·min vs. 2.49 ± 0.30 in LND, 3.12 ±
0.41 vs. 3.02 ± 0.42 in OND, and 2.73 ± 0.31
vs. 2.64 ± 0.33 in diabetic groups). There was also no
effect of wortmannin on total activity of GS after insulin stimulation.
The same treatment caused a significant reduction of basal activity of
GS, measured at 0.1 mM G-6-P, reflected in a
reduction of GS FV in all three groups (diabetic: 0.042 ± 0.011;
OND: 0.047 ± 0.012; LND: 0.068 ± 0.011). This concentration
of wortmannin inhibited insulin stimulation of GS FV equally in
nondiabetic (71% and 80% inhibition of insulin effect in lean and
obese subjects, respectively) and diabetic (82%) muscle cells (Fig. 1). These results indicate that wortmannin does not affect GS activity
directly but rather inhibits the activation state of the enzyme by
impairing signal transduction through inhibition of PI 3-kinase.
Treatment of cells with a second, structurally unrelated inhibitor of
PI 3-kinase inhibitor, LY 294002 (10 µM),
resulted in inhibition of insulin activation of GS similar (80–90%)
to that caused by wortmannin.
Incubation of muscle cells with a lower (25 nM) wortmannin
dose only modestly decreased basal or insulin-stimulated GS activity in
the LND or obese groups (0.147 ± 0.027 and 0.16 ± 0.015,
respectively) but significantly diminished insulin-stimulated GS FV
in T2D cells (0.068 ± 0.010). There was also a significantly
greater reduction of the insulin increment of synthase FV in muscle
cells of diabetic (71 ± 11%), compared with lean and OND
subjects (24 ± 7 and 33 ± 14, Fig. 1).
Impaired insulin activation of PI 3-kinase but normal protein expression in muscle cells of T2D subjects
To investigate whether differences in the effects of wormannin and
LY 294002 between diabetic and nondiabetic cells could be attributable
to differences in activities and expression of PI 3-kinase proteins, we
measured IRS-1associated PI 3-kinase activity and protein
abundance of the p85- and p110ß-subunits of PI 3-kinase.
Incubation of nondiabetic muscle cells, for 30 min, with insulin
increased IRS-1-associated PI 3-kinase activity by more than 7-fold
(Fig. 2). In cells of T2D subjects,
insulin activation of PI 3-kinase activity was significantly diminished
(4.3 ± 0.7 vs. 7.2 ± 0.8-fold, compared with
LND, P < 0.05). The response of PI 3-kinase to insulin
was intermediate in cells of OND subjects (5.7 ± 0.9-fold, Fig. 2B).
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and p110ß
proteins was similar in nondiabetic and diabetic cultures (Fig. 3, A and C). Because insulin activation
of PI 3kinase can involve binding to IRS-1, we also measured IRS-1
protein levels in the same cultures of nondiabetic and diabetic
subjects. There were no significant differences in IRS-1 protein levels
between nondiabetic and diabetic subjects (Fig. 3, B and C).
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Possible involvement of Akt, MAPK, and p70 S6 kinase in insulin signaling to GS
Because Akt, MAPK, and p70 S6 kinase may all be affected by wortmannin and lie downstream of PI 3-kinase (19, 40, 41), we investigated the activation and expression of these kinases in muscle cell cultures of diabetic subjects, in an attempt to reveal intermediate steps of insulin signaling leading to activation of GS.
Insulin (30 nM) treatment of muscle cells resulted in the
appearance of a phosphorylated form of Akt (Fig. 4A). Insulin-induced phosphorylation of
Akt was blocked by preincubation of cells with wortmannin (50
nM) or LY294002 (10 µM). Rapamycin and/or
PD098059 inhibited neither PI 3kinase activity (not shown) nor Akt
phosphorylation (Fig. 4C), suggesting that PI 3-kinase, but not p70 S6
kinase or MAPK, is involved in the insulin signaling pathway leading to
Akt phosphorylation. Unlike the situation for IRS-1 associated PI
3-kinase activation, insulin-stimulated phosphorylation of Akt and Akt
protein expression were similar in muscle cells from all groups (Fig. 4, A and B).
|
The levels of phosphorylated and total MAPK1 and MAPK2 were similar in
cells from nondiabetic and T2D subjects (Fig. 5A) in both the absence and presence of
insulin.
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).
Wortmannin treatment (50 nM) also reduced (70%
inhibition) insulin stimulation of MAPK phosphorylation.
Insulin-stimulated GS FV was reduced (diabetic: 0.077 ± 0.019,
nondiabetic obese: 0.086 ± 0.016, LND: 0.133 ± 0.029, all
P < 0.05 vs. control), and the insulin
increment was diminished by approximately 30–40% after PD098059
treatment in cells of diabetic and nondiabetic subjects (Fig. 6).
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) in a time-dependent manner, with the maximum
effect after 30 min of incubation (a time course similar to that of GS
activation). Preincubation of cells with 50 nM rapamycin
completely prevented the appearance of the phosphorylated p70 S6 kinase
(Fig. 5D). This concentration of rapamycin has been previously shown to
inhibit (by >60%) insulin stimulation of p70 S6 kinase activity under
similar conditions (24, 26, 30). Rapamycin (50
nM) treatment had no influence on insulin-induced
MAPK phosphorylation, Akt phosphorylation, or PI 3-kinase activation
(not shown).
There were no significant differences in insulin-stimulated Thr389
phosphorylation of p70 S6 kinase between diabetic and nondiabetic
groups (Fig. 5C). Protein expression of p70 S6 kinase was also similar
in all subjects (Fig. 5C). Rapamycin treatment was without significant
effect on basal GS activity in all groups. Insulin-stimulated GS FV was
reduced after incubation with rapamycin in OND subjects (0.110 ±
0.021, P < 0.01), which resulted in a decrease in the
insulin increment of GS FV (Fig. 6). In LND and diabetic subjects, the
effect of rapamycin on insulin-stimulated GS was less pronounced (Fig. 6) and not statistically significant (0.152 ± 0.029,
P = 0.08 by paired analysis in LND cells; 0.077 ±
0.019, P = 0.08 in diabetic cells). Activities of GS
measured at 10 mM of G-6-P, an indicator of the
fully activated enzyme, were unaffected by treatment in both
nondiabetic (2.67 ± 0.25 vs. 2.92 ± 0.26 nmol
UDPG/min·mg protein, P = not significant) and
diabetic cultures (2.74 ± 0.18 vs. 2.76 ± 0.18,
P = not significant), indicating that the effects of
rapamycin did not involve direct inhibition of synthase activity.
Discussion
Both the activity of GS and its responsiveness to insulin have been found to be impaired in skeletal muscle biopsy samples of T2D subjects (8, 33), differences that may play a major role in the development of glucose intolerance and insulin resistance. It is possible that these defects involve both acquired and genetic components, considering that impairments of basal and insulin-stimulated activity of GS are retained in cultured skeletal muscle cells obtained from T2D subjects cultured under normoglycemic and -insulinemic conditions (9).
With regard to possible insulin stimulation of GS, potentially the most significant signaling pathways are the ras/MAPK/p90rsk, mTOR/p70 S6 kinase, and PI 3-kinase/protein kinase B (Akt) pathways. There is considerable cross-talk and overlap of these pathways, because activity of the ras/MAPK and mTOR/p70 S6 kinase pathways can be controlled in both a PI 3-kinase-dependent and -independent manner (19, 40, 41, 42, 43). Evidence has accumulated to support a key role for PI 3-kinase in mediating insulin action on glucose transport and GS (44, 45). Akt has been designated as a crucial downstream effector of PI 3-kinase (13), but the evidence is mixed concerning whether Akt is essential for control of glucose metabolism (46). Further complicating the question of insulin signaling to GS are discrepancies in the literature, suggesting that the relative importance of different pathways may vary with the species and tissue studied (10, 14, 21, 24, 47). Possible cellular specificity of insulin signaling is one reason why it is important to investigate this question in systems closely resembling the behavior of skeletal muscle, the most significant insulin target tissue in humans. We have employed differentiated myotubes, which display signaling pathways and effector systems similar to those that exist in mature skeletal muscle. Studies in human muscle cultures are also relevant to the in vivo situation, because cultured muscle cells from T2D subjects manifest defects in GS activity and insulin action that are highly reflective of the behavior measured in vivo and in muscle biopsies (33).
Though attention has properly focused on insulin regulation of GS, it is important to emphasize that synthase activity in the absence of added insulin, the basal state, is also the result of a balance between enzyme phosphorylation and dephosphorylation. In all groups, wortmannin, but not PD098059 or rapamycin, was able to significantly decrease basal FV, indicating that insulin-independent control of GS also involves PI 3-kinase-dependent processes. Because basal skeletal muscle GS activity is reduced in type 2 diabetes, both in biopsies (8) and cultured myotubes (9), these events involving PI 3-kinase take on added importance.
The current data obtained in differentiated human myotubes are also
supportive of a central role for PI 3-kinase in insulin stimulation of
GS. The primary objective of the current work was to identify which
steps in insulin signaling might be altered in diabetic muscle,
contributing to impaired insulin action. There were two significant
differences observed between muscle cells from T2D subjects and those
from nondiabetic subjects: a greater sensitivity to wortmannin for
inhibition of insulin signaling to GS (Fig. 1), and impaired insulin
stimulation of IRS-1-associated PI 3-kinase activity (Fig. 2).
Impairments in insulin-stimulated PI 3kinase activity in diabetic
muscle biopsies have also been reported (29, 31). Some of
those same subjects characterized in one of those earlier studies
(29) provided tissue for muscle cultures analyzed in the
current study. Consideration of the in vivo and in
vitro data reveals that the impairment of insulin stimulation of
PI 3-kinase was not resolved upon culture of diabetic muscle cells in a
normal metabolic milieu, suggesting that the step of activation of PI
3-kinase in diabetic muscle cells may be intrinsically defective and
could lead to impairments in insulin activation of GS.
A greater sensitivity of diabetic muscle to wortmannin, for insulin
stimulation of GS FV, could be attributable to defects in protein
expression of the components of the PI 3-kinase signaling system.
However, expression of key proteins in this signaling pathway,
IRS-1, the p85 regulatory subunit, and the p110ß catalytic
subunit, did not differ significantly between nondiabetic and diabetic
muscle cells, in agreement with normal expression in muscle strips from
diabetic subjects (31). Thus, impairment in PI 3-kinase
activation occurs despite normal expression of proteins involved in
this process, indicating a reduction in the function of one or more of
the components of this system. Possible mechanisms for impaired action
could include loss of function mutations or posttranslational
modification of these proteins. Indeed, mutations have been identified
in p85 (48) and IRS-1 (49) but not p110ß
(50). Alternatively, the role of compartmentalization in
the organization of insulin signaling has been a topic of recent
scrutiny (51), and it may be that defective signaling in
diabetic muscle involves mistargeting of key proteins. Impaired insulin
stimulation of IRS-1-associated PI 3-kinase could also involve more
proximal signaling events, such as insulin receptor kinase activity, a
question currently under investigation.
We (29) and others (52) have shown that, despite defects in PI 3-kinase activation, at physiological insulin levels, insulin stimulation of Akt phosphorylation is essentially normal in muscle from obese T2D subjects, the same behavior as determined in cultured muscle cells. This second report (52) did find a reduction in insulin activation of Akt activity but only at supraphysiological insulin levels. Full activation of Akt, despite impaired PI 3-kinase, could occur through several mechanisms. One would be that, at least in human skeletal muscle, only a portion of available PI 3-kinase activity is required to fully phosphorylate/activate Akt. Another possibility is that the measurement of total Akt phosphorylation, as performed in the current report, may obscure changes in a smaller pool of Akt directly linked to GS. The fact that insulin stimulation of GS remains impaired in diabetic muscle cells, even in the presence of intact Akt, indicates that other downstream pathways may play a role in signal transduction to GS.
In addition to differences observed in diabetic muscle cells, certain
of the current findings in differentiated human myotubes differ, at
least in a quantitative manner, from results in other systems. For
example, we observed that PD098059 is able to block a significant
portion (30–40%) of the insulin effect on GS (Fig. 6), indicating
that, in differentiated human myotubes, MAPK can significantly
influence GS activity. Meanwhile, in other systems, MAPK plays little
or no role in the metabolic responses to insulin (12, 20, 22, 24). Interestingly, whereas only a minor portion (20%) of
the insulin effect on GS may occur through p70 S6 kinase, in muscle
cells from the OND group, the effect of rapamycin was significantly
greater (40%) than in LND subjects. Thus, this pathway may be
relatively more important for insulin activation of GS in muscle of
obese subjects and possibly contribute to the insulin insensitivity of
GS in obesity. Additionally, our results indicate that the expression
and insulin responsiveness for phosphorylation of p70 S6 kinase and
MAPK are essentially normal in diabetic muscle cells. In agreement with
our results, expression and insulin-induced phosphorylation of MAPK
have been found to be normal in isolated skeletal muscle from T2D
patients (31). The impact of type 2 diabetes on p70 S6
kinase has not been previously studied in human muscle, especially
in vivo, so the current work is a first report that this
signaling enzyme is intrinsically intact in diabetic muscle.
Our data are in agreement with a proposed model for signal transduction pathways in which stimulation of PI 3-kinase is an early event in insulin signaling leading to activation of at least three regulatory enzymes and pathways: Akt, p70 S6 kinase, and MAPK. The last two are minor pathways leading to GS stimulation, given that complete inhibition of the enzymes or their activation caused only modest inhibition of synthase activation by insulin.
The current studies reveal several features of insulin signaling in T2D skeletal muscle that might not be apparent from studies in other systems. The first is that impairments in PI 3-kinase seen in vivo are not reversed in culture and probably represent an intrinsic defect. Additionally, this signaling defect is not a generalized one, because insulin signaling to p70 S6 kinase, MAPK, and Akt are all intact. Whatever the ultimate mechanisms, it seems that insulin resistance in type 2 diabetes involves multiple insulin signal transduction pathways in addition to defects in final effector systems.
Acknowledgments
Footnotes
This work was supported by the Medical Research Service, Department of Veterans Affairs, the Veterans San Diego Healthcare System, a Career Development Award from American Diabetes Association (to S.E.N.), by funds from the Whittier Institute for Diabetes, and by Grant M01-RR-00827 from the General Clinical Research Centers program of the National Center for Research Resources, NIH.
Abbreviations: ERK, Extracellular regulated kinase; FV, fractional velocity; GDR, glucose disposal rates; GS, glycogen synthase; GSK3, GS kinase 3; IC50, concentration to attain 50% inhibition; LND, lean nondiabetic; OND, obese nondiabetic; PI 3-kinase, phosphatidylinositol 3-kinase; PP1-G, glycogen-associated protein phosphatase-1; T2D, type 2 diabetic subjects; UDP, uridine diphospho.
Received February 1, 2001.
Accepted May 30, 2001.
References
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E. A. McIntyre, R. Halse, S. J. Yeaman, and M. Walker Cultured Muscle Cells from Insulin-Resistant Type 2 Diabetes Patients Have Impaired Insulin, but Normal 5-Amino-4-Imidazolecarboxamide Riboside-Stimulated, Glucose Uptake J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3440 - 3448. [Abstract] [Full Text] [PDF]
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 K. Bouzakri, M. Roques, P. Gual, S. Espinosa, F. Guebre-Egziabher, J.-P. Riou, M. Laville, Y. Le Marchand-Brustel, J.-F. Tanti, and H. Vidal Reduced Activation of Phosphatidylinositol-3 Kinase and Increased Serine 636 Phosphorylation of Insulin Receptor Substrate-1 in Primary Culture of Skeletal Muscle Cells From Patients With Type 2 Diabetes Diabetes, June 1, 2003; 52(6): 1319 - 1325. [Abstract] [Full Text] [PDF]
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K. Hojlund, P. Staehr, B. F. Hansen, K. A. Green, D. G. Hardie, E. A. Richter, H. Beck-Nielsen, and J. F.P. Wojtaszewski Increased Phosphorylation of Skeletal Muscle Glycogen Synthase at NH2-Terminal Sites During Physiological Hyperinsulinemia in Type 2 Diabetes Diabetes, June 1, 2003; 52(6): 1393 - 1402. [Abstract] [Full Text] [PDF]
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D. Dietze, M. Koenen, K. Rohrig, H. Horikoshi, H. Hauner, and J. Eckel Impairment of Insulin Signaling in Human Skeletal Muscle Cells by Co-Culture With Human Adipocytes Diabetes, August 1, 2002; 51(8): 2369 - 2376. [Abstract] [Full Text] [PDF]
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S. E. Nikoulina, T. P. Ciaraldi, S. Mudaliar, L. Carter, K. Johnson, and R. R. Henry Inhibition of Glycogen Synthase Kinase 3 Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle Diabetes, July 1, 2002; 51(7): 2190 - 2198. [Abstract] [Full Text] [PDF]
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, P < 0.05,
compared with the same condition in LND cultures.
