Department of Biochemistry, School of Medicine, University of Patras, 26110 Patras, Greece
Address all correspondence and requests for reprints to: Athanasios G. Papavassiliou, M.D., Ph.D., Professor and Chairman, Department of Biochemistry, School of Medicine, University of Patras, GR-26110 Patras, Greece. E-mail: papavas{at}med.upatras.gr
ABSTRACT
Insulin resistance, the failure to respond to normal circulating
concentrations of insulin, is a common state associated with obesity,
aging, and a sedentary lifestyle. Compelling evidence implicates TNF
as the cause and link between obesity and insulin resistance. Serine
phosphorylation of insulin receptor substrate-1 seems prominent among
the mechanisms of TNF
-induced insulin resistance. Recent advances
indicate that serine kinases may phosphorylate and thus inhibit the
tyrosine phosphorylation of insulin receptor substrate-1,
revealing an integration point of TNF
and insulin signaling
pathways. Selective targeting of the molecular scenery whereby this key
phosphorylation occurs/operates represents a rich area for the
development of rationally designed new antidiabetic drugs. In relation
to efficacy and side effects, this prospect should permit a more
precise and perhaps individualized approach to therapeutic
intervention, allowing clinicians to focus the attack where the problem
lies.
TYPE 2 DIABETES mellitus (T2DM) is a prevalent cause of morbidity and mortality, mainly through its long-term cardiovascular complications. Despite decades of intense investigation, its pathogenesis remains incompletely understood. Clinical T2DM is thought to ensue when insulin production by the pancreas fails to compensate for the insulin resistance manifested by the target tissues of insulin. In diabetic patients, skeletal muscle, adipose tissue, and the liver display a lower than normal response to insulin action. Insulin resistance is believed to be the earliest and dominant metabolic defect in T2DM and is central to the pathogenesis of hypertension, hyperlipidemia, atherosclerosis, and other constituents of the metabolic syndrome. T2DM is a multifactorial disease with a strong genetic component. The major predisposing environmental factor is obesity, which is virtually always associated with insulin resistance (1).
During the past decade TNF, a cytokine, has been implicated as a
link between obesity and insulin resistance. Data have come from three
lines of evidence (2): 1) association studies, in which
TNF
production by animal and human adipose and muscle tissues has
been correlated with obesity and indices of insulin resistance; 2)
in vivo experiments, which utilize TNF
infusion and
neutralization in animals and humans; and 3) transgenic mice, in which
the cytokine or its receptors have been knocked out. TNF
is not the
sole mediator of insulin resistance in obesity; other candidates
include FFAs, leptin, hyperinsulinemia, and the newly discovered
hormone, resistin (3). TNF
is expressed as a
transmembrane protein that is cleaved to release a soluble cytokine.
Muscle- and fat-derived TNF
may act on target cells in an endocrine,
paracrine, or autocrine manner, and both soluble and transmembrane
forms can associate with TNF
receptors (4). Receptors
are devoid of enzymatic activity but serve as docking molecules for
other signaling proteins that induce both apoptotic and antiapoptotic
signals.
Multiple, not mutually exclusive, mechanisms have been proposed to
account for TNF-induced insulin resistance in obesity
(4). These include elevation of plasma FFAs due to its
lipolytic action, down-regulation of insulin-sensitive glucose
transporter (GLUT4) translocation to the plasma membrane, antagonism of
the PPAR
pathway, interplay with leptin and resistin, and, most
interesting, direct interference with insulins signal transduction
pathway.
Insulin binding to the -subunits of its heterotetrameric membrane
receptor triggers the tyrosine kinase (insulin receptor kinase, IRK)
activity of the ß-subunits, which transphosphorylate themselves and
tyrosine-phosphorylate endogenous substrates, including Shc and
insulin receptor substrates (IRSs). IRSs are related protein substrates
of IRK, with a highly conserved N terminus containing a PH domain and a
phospho-Tyr-binding (PTB) domain, and a poorly conserved C terminus
with several Tyr phosphorylation motifs (5). At least four
IRS isoforms occur in mammals: IRS-1 and IRS-2 are widely expressed,
whereas IRS-3 is restricted to adipose tissue and IRS-4 is expressed in
the thymus, brain, and kidney. To delineate the functional roles of IRS
isoforms, IRS and insulin receptor (IR) genes have been disrupted to
create homozygous null-mice and various combinations of homozygous-null
and/or compound heterozygous (5, 6). Moreover, the
in vivo impact of IRS-1 vs. IRS-2 gene disruption
on carbohydrate and lipid metabolism has been assessed by euglycemic
hyperinsulinemic clamps (7). These studies suggest that
IRS isoforms have tissue-specific roles: IRS-3 and IRS-4 appear to have
minor or redundant roles in insulin signaling. IRS-1 is the most
important isoform in muscle, whereas IRS-2 has an impact on liver,
muscle, and adipose tissue. Because of exciting new insights into the
molecular machinery that fine tunes IRS-1 function, the scope of this
presentation will be restricted to the latter.
After Tyr phosphorylation IRS-1 serves as a docking protein for a
number of effector molecules bearing the SH2 domain, such as Grb2, Syp,
Nck, and the regulatory subunit of PI3K, p85. p85 binding to IRS-1
stimulates the PI-3K activity of the p110 catalytic subunit, as well as
downstream signaling proteins. Phosphorylated Shc and IRS-1 bind to
Grb2 and mediate p21ras GTP-loading via the
guanyl nucleotide exchange factor, SOS. Active
p21ras associates with and activates the Raf-1
kinase (a MAPK kinase kinase, MAPKKK), which phosphorylates and
activates MEK-1 (a MAPK kinase, MAPKK), which in turn phosphorylates
and activates ERK, a member of the MAPK family of signaling enzymes.
Activation of the MAPK cascade is associated mostly with
transcriptional and mitogenic effects of insulin, while the PI3K
pathway is generally engaged with the hormones metabolic effects
(Fig. 1).
|
Increased serine phosphorylation of IRS-1 is the proposed molecular
mechanism by which TNF inhibits insulin signaling (4).
In cultured fat cells TNF
induces Ser phosphorylation of IRS-1,
which subsequently inhibits IRK activity in vitro. The
presence of IRS-1 is required for TNF
-induced inhibition of insulin
signaling in intact cells. Furthermore, an inhibitory form of IRS-1 is
also present in adipose tissue and skeletal muscle of obese
fa/fa rats. In cultured hepatoma cells, TNF
also induces
Ser phosphorylation of IRS-1 and decreases its association with PI3K,
but does not affect IRK activity. Thus, TNF
-induced Ser
phosphorylation of IRS-1 is central to inhibition of insulin
signaling, but the precise inhibitory function of
Ser-phosphorylated IRS-1 may depend on cell type. In hepatoma
cells it has been shown that TNF
-induced Ser phosphorylation of
IRS-1 impairs its interaction with the juxtamembrane (JM) domain of IR
and may thus render IRS-1 a poorer substrate for IRK (8).
Alternatively, Ser-phosphorylated IRS-1 may recruit unknown signaling
molecules that sterically hamper its interaction with IR and/or inhibit
IRK activity.
Increased serine/threonine phosphorylation of IRS-1 is a common finding
in insulin resistance and T2DM. Numerous agents that induce insulin
resistance, such as TNF and other cytokines, okadaic acid,
platelet-derived growth factor, angiotensin II, hyperglycemia, and
hyperinsulinemia, all increase IRS-1 phospho-Ser/phospho-Thr content.
Some, including TNF
, are activators of Ser/Thr kinases, while others
are inhibitors of Ser/Thr phosphatases. Interestingly, IRS-1 is Ser
phosphorylated at the basal level, and this phosphorylation is
augmented after insulin stimulation. Therefore, Ser/Thr phosphorylation
of IRS-1 emerges as a negative feedback mechanism in normal insulin
signaling, which is also employed by various factors that counteract
insulin action (9). The kinetics of insulin-induced IRS-1
Ser/Thr phosphorylation, as compared with signal-propagating Tyr
phosphorylation, is compatible with this notion. In a parallel way,
Ser/Thr phosphorylation of SOS after prolonged insulin stimulation
leads to dissociation of the Grb2/SOS complex. The MAPK pathway is
subsequently inactivated as GTP-p21ras is
hydrolyzed to the inactive GDP-bound form (10).
However, not all IRS-1 Ser phosphorylation is inhibitory: Protein kinase B (PKB/Akt) lies downstream of PI3K and is phosphorylated and activated by PI3K-dependent kinase (PDK1). After insulin stimulation, PKB has been shown to phosphorylate Ser residues within and adjacent to the PTB domain of IRS-1 (Ser265, Ser302, Ser325, and Ser358 in mouse IRS-1) (11). Phosphorylation at these sites protects IRS-1 from the action of Tyr phosphatases and maintains the substrate in its signal-propagating, Tyr-phosphorylated form. Therefore, Ser phosphorylation of IRS-1 at specific residues could be part of a positive feedback loop, in which PKB is the key switch. Insulin-activated kinases involved in negative feedback loops are expected to lie downstream or independent from PKB. Theoretically, however, a kinase upstream of PKB might also be responsible, at least in part, as it may well be that it is the vigor and rhythm of protein-to-protein cross-talk that determines the resultant outcome on a target molecule rather than the sequence of the molecules in the cascade. The action of the positive and negative circuits must nonetheless be temporally coordinated in a way that permits the propagation and amplification of the insulin cascade by the positive loop, before the negative loop predominates to attenuate the signal.
The identification of the insulin-dependent kinase or kinases that
mediate the inhibitory Ser/Thr phosphorylation of IRS-1 and of their
target residues is an area of intensive investigation (reviewed in Ref.
12). This identification is important because it may
permit the development of rationally designed pharmaceutical agents
that will interfere with the molecular events involved in insulin
resistance. It is, however, a fierce task, given that IRS-1 contains
more than 30 potential Ser/Thr phosphorylation sites nested within
sequences that conform to various kinase motifs, including PKA, PKC,
MAPK, casein kinase II, Cdc2 kinase, and PKB/Akt. At least some of the
unknown kinases, should there be more than one, are downstream
effectors of PI3K, because their action is sensitive to wortmannin, a
specific PI3K inhibitor (11). Therefore, the search is for
a kinase downstream of PI3K other than positively acting PKB. MEK-1 and
p38 MAPK are unlikely to be involved, because they are not wortmannin
sensitive and their inhibitors do not interrupt the feedback loop.
Inhibitors of PKC isoforms , ß,
,
,
, and µ were also
ineffective (11). Glycogen synthase kinase-3 is capable of
phosphorylating IRS-1 and impairing the insulin signal, but is not
likely to be the kinase in question because insulin stimulation
inhibits glycogen synthase kinase-3 (13). PI3K-dependent
kinases (PDKs) are also potential candidates, but they are located
upstream of PKB. The same holds true for PI3K itself, which has been
shown to stimulate IRS-1 Ser phosphorylation and inhibit
IRS-1-associated PI3K activity when a membrane-targeted form is
expressed in 3T3-L1 adipocytes (14). PKB isoforms ß and
cannot be ruled out, but are also unlikely candidates because their
substrate selectivity is similar to that of PKB
.
The chase for the insulin-induced kinase that inhibits IRS-1 has not,
in fact, been totally unfruitful. Tetradecanoylphorbol acetate, a
potent activator of several PKC isoforms, inhibits the interaction of
IRS-1 with the JM domain of IR and the ability of insulin to
phosphorylate IRSs (8). PKC modulation of IRS-1 Tyr
phosphorylation requires Ser612 (of rat IRS-1)
(15), and this effect may be mediated by MAPK
(16). Among PKC isoforms, atypical members such as PKC,
-
, and -
, are prominent candidates. Indeed, PKC
is a
downstream effector of PI3K that Ser phosphorylates IRS-1 and impairs
its Tyr phosphorylation at specific residues
(Tyr612 and Tyr632 in human
IRS-1) and its ability to activate PI3K in response to insulin
(17). The specific Ser-phosphorylation sites of PKC
have not yet been identified. Other likely candidates are protein
kinases p70 kDa S6 and mTOR (mammalian target of rapamycin), which are
downstream of PDK1 and PKB. The PI3K/PKB pathway phosphorylates IRS-1
at residues Ser632, Ser662,
and Ser731 (in rat IRS-1) and inhibits its
ability to bind PI3K and activate PKB; this effect is sensitive to
rapamycin, an inhibitor of mTOR (18, 19). Interestingly, a
rapamycin-dependent pathway also leads to IRS-1 degradation by the
proteasome (20). This effect is presumably regulated by
the balance of Ser vs. Tyr phosphorylation of IRS-1
(21). However, the Ser phosphorylation sites that mediate
the degradation are different to those that functionally inhibit IRS-1,
although both pathways are mediated by mTOR. This implies a divergence
of the two pathways downstream of mTOR with the possible recruitment of
different kinases.
Previous evidence suggested that TNF-induced activation of the c-Jun
N-terminal kinase (JNK) leads to phosphorylation of
Ser307 in IRS-1, and that this may mediate, at
least in part, the inhibitory effect of TNF
on insulin
signaling (22). JNK is a member of the MAPK family,
activated in the general MAPKKK
MAPKK
MAPK scheme. JNK is also
termed stress-activated protein kinase because it responds to various
stress signals, whereas the ERK pathway is responsive to growth and
differentiation stimuli. Using CHO cells expressing IR and
IRS-1, it has been demonstrated that JNK associates with the C terminus
of IRS-1 in quiescent cells and phosphorylates it when activated
(22). Phosphopeptide mapping and mutational analyses
identified Ser307, a residue conserved in all
IRS-1 homologs, as a major site of JNK phosphorylation. Moreover, it
was shown that in myeloid 32D cells and in 293 cells, a
Ser307-to-Ala mutation abolishes the inhibitory
effect of TNF
on insulin-induced IRS-1 Tyr phosphorylation. There
was no proof that JNK is the endogenous kinase that mediates
obesity-linked, TNF
-associated insulin resistance in muscle and
adipose tissue. However, JNK is a downstream effector of TNF
(Fig. 1
) and has also been implicated as both a mediator and negative
modulator of insulin signaling. Thus, the identification of JNK as a
kinase that phosphorylates IRS-1, and of the phosphorylated residue,
Ser307, as a TNF
-induced phosphorylation
target revealed JNK as a converging point in insulin signaling, which
mediates both downstream propagation of signals as well as
desensitization of the pathway by TNF
and insulin itself. As such,
JNK would qualify as a target for insulin resistance-directed drug
development. However, this inhibition might not be without undesirable
consequences, because JNK is ubiquitously expressed and is involved in
a variety of housekeeping functions including apoptosis, as well as
some of the metabolic and transcriptional effects of insulin.
Very recently, the same research group has studied IRS-1
Ser307 phosphorylation in response to
insulin/IGF-I and TNF in 3T3 L1 preadipocytes and adipocytes, as
well as Ser307 phosphorylation in response to
insulin in skeletal muscle of mice, rats, and humans (23).
They reverified that Ser307 is phosphorylated by
these stimuli, leading to impairment of insulin signaling. Importantly,
using selective inhibitors, they identified distinct pathways that are
activated in response to insulin and TNF
and converge on IRS-1
Ser307. Phosphorylation in response to insulin is
sensitive to wortmannin or LY294002, which implies that it is mediated
by the PI3K pathway, whereas phosphorylation in response to TNF
is
sensitive to PD98059 and thus appears to be MEK-1 dependent (Fig. 1
).
Surprisingly, JNK was not found to participate in
Ser307 phosphorylation. These new data have two
important implications. First, Ser307 emerges as
a possible hallmark of insulin resistance in biologically important
cells and tissues. Second, because different kinases are activated by
distinct signals and converge on a single residue to promote insulin
resistance, therapeutic targeting can be directed away from the
unrealistic goal of kinase inhibition and toward the specific
prevention or reversal of Ser307 phosphorylation,
or against its effect on the insulin signaling cascade.
The demonstration that TNF Ser-phosphorylates IRS-1 via a
PI3K-independent pathway contrasts somewhat with the findings of Ozes
et al. (24). Using NIH 3T3 cells, 293 embryonic
kidney cells, and C3H 10T1/2 C18 myoblasts, these investigators
demonstrate that TNF
Ser-phosphorylates IRS-1 through the
PI3K/PKA/mTOR pathway and inhibits insulin-induced Tyr
phosphorylation of IRS-1. PTEN, a human tumor suppressor gene
and phospholipid phosphatase that inhibits PI3K and PKB pathways, is
shown to antagonize TNF
insulin resistance. Their analysis
identified residues other than Ser307 as the
important phosphorylation sites, i.e. Ser636 and
Ser639. It is conceivable that TNF
may activate both the
PI3K and MAPK pathways and each pathway may phosphorylate different
serines on IRS-1. Thus, an important question regarding the candidate
kinases and residues that inhibit IRS-1 function or promote its
degradation in response to insulin or external stimuli, is that of the
relative (and hierarchical?) contribution of specific kinase/residue
phosphorylation to the overall insulin resistance. To this end, we note
that studies employing tissues, animals, and human subjects
(23) may be preferential to in vitro
(cell-line) experiments (24), because they are more likely
to identify pathways and processes that closely resemble the in
vivo situation and thereby unveil clinically promising
pharmaceutical targets.
The precise molecular mechanism of insulin signaling inhibition by
IRS-1 Ser307 phosphorylation is presently
unknown. A prominent candidate mechanism is the inhibition of
functional interaction between phosphorylated IRS-1 and IR.
Ser307 is adjacent to the PTB domain of IRS-1,
which lies C-terminally to the PH domain in the N terminus of IRS-1.
The PH domain targets IRS-1 to the plasma membrane and allows the PTB
domain to bind to a phosphorylated Tyr in the JM domain of IR and form
a functional contact between the two proteins (Fig. 1)
(5). It is possible that Ser307
phosphorylation impairs this functional interaction and thus inhibits
insulin signaling (8). An alternative mechanism may
involve the recruitment of inhibitory molecules by
phospho-Ser307, such as Tyr phosphatases or other
proteins that interfere with insulin signaling.
The crystal structure of IRS-1 PTB, alone and complexed with the JM region of IR, is known (25). Moreover, the three-dimensional structure of the PH-PTB targeting region of IRS-1 has recently been solved (26). We propose that this knowledge may suffice for the purpose of drug development, through high-throughput screening of available large-compound libraries or through structure-based (de novo) ligand design methodologies (27). The aim would be to search for or design small-molecule drugs that will bind to the N terminus of IRS-1 with high affinity and extreme selectivity and stabilize (trap) the three-dimensional conformation of the PH-PTB region in its IR-bound form, so that it will not be influenced by the effects of Ser307 phosphorylation. A different strategy would be to discover organic ligands that bind to or in the vicinity of Ser307 and prevent its phosphorylation by masking it from cognate kinases, or minimize its deleterious effects. A major challenge of these rational approaches will entail incorporating IRS-1 tissue distribution, cellular topography, and isotype selectivity, as well as changes in macromolecular interaction profile elicited by Ser307 phosphorylation.
Undoubtedly, the molecular events that lead to insulin resistance are
only partly understood, and it is well recognized that multiple
mechanisms are involved. However, development of drugs need not and
cannot await detailed elucidation of signaling pathways. Currently
available insulin-sensitizing drugs are the thiazolidinediones,
activators of the PPAR transcription factors, which increase insulin
sensitivity possibly through down-regulation of the adipocyte-derived
resistin (3). However, thiazolidinediones may have
untoward side effects in tissues other than fat expressing PPAR
(e.g. colorectal carcinoma and polyps, atherosclerosis), and
it remains unclear whether they truly tackle the underlying metabolic
defects. The identification of IRS-1 Ser307 as an
in vivo phosphorylation site with pivotal importance may
provide a promising pharmaceutical target in the regimen of treatment
of T2DM and its related syndromes. Similar insights into the IRS-2
molecule, which is crucial also for ß-cell compensation in the face
of insulin resistance (7), might lead to the development
of rationally designed restorers of both the peripheral insulin
responsiveness and ß-cell inadequacy that characterize this
devastating disease.
FOOTNOTES
Abbreviations: IR, Insulin receptor; IRK, insulin receptor kinase; IRS, insulin receptor substrate; JM, juxtamembrane; JNK, c-Jun N-terminal kinase; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase; MEK, MAPK kinase; mTOR, mammalian target of rapamycin; PDK, PI3K-dependent kinase; PKB, protein kinase B; PTB, phospho-Tyr binding; T2DM, type 2 diabetes mellitus.
Received for publication June 12, 2001. Accepted for publication July 23, 2001.
REFERENCES