Issra Rashed
Makinen Laboratory
Protein tyrosine phosphatase 1B (PTP-1B) helps regulate several cellular processes,
including the deactivation of both the insulin and epidermal growth factor receptors. PTP-1B is
therefore the object of many inhibitor design studies; however, attempts to find or design potent
and specific PTP-1B inhibitors have been quite difficult because the active sites and structural
motifs of the known PTPs are heavily conserved. Recently,
bis(acetylacetonato)oxovanadium(IV) [VO(acac)2] has been shown to uncompetitively inhibit
PTP-1B in the presence of a physiologically relevant, phosphotyrosine (pTyr) containing 12
amino acid substrate, DADEpYLIPQQG. An uncompetitive inhibitor binds to only the enzymesubstrate (ES) complex, as
opposed to a competitive or
non-competitive inhibitor
that binds to the enzyme
alone. Despite the sequential
and structural homology of
the 100 known PTPs, these
phosphatases exhibit nonredundant roles such that
each PTP has unique ES
complexes.
The object of my studies is to characterize and explore the interaction of VO(acac)2 with
the PTP-1B- DADEpYLIPQQG enzyme-substrate complex, to evaluate the specificity of
VO(acac)2 to PTP-1B in the presence of other substrates, and also to examine the effect of
VO(acac)2 on other PTPs.
572
Surface plasmon resonance: towards an understanding of the
mechanisms of biological molecular recognition
James M McDonnell
With the introduction of new instruments and improved sensor
chip chemistries, surface plasmon resonance (SPR) is finding
new applications for molecular interaction studies. Easy access
to high-quality kinetic and thermodynamic data for
macromolecular binding events is providing insights into the
fundamental mechanisms of molecular recognition. Progress is
being made to allow larger-scale interaction studies. In
addition, combining SPR with other analytical methods is
enabling SPR-based analysis of interaction proteomics.
biological systems, and recently there has been an expansion of applications into previously rare areas.
Addresses
The Laboratory of Molecular Biophysics, Department of Biochemistry,
University of Oxford, South Parks Road, Oxford OX1 3QU, UK;
e-mail: [email protected]
SPR instrumentation and biosensor chips
Current Opinion in Chemical Biology 2001, 5:572–577
1367-5931/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
CPWR couple plasmon-waveguide resonance
EGF
epidermal growth factor
IgE
immunoglobulin E
MHC
major histocompatibility complex
SPR
surface plasmon resonance
Introduction
The biological functions of most macromolecules depend
on their ability to interact with other molecules. In the
post-genomic era, one of the greatest challenges facing the
chemical biology community is a complete description of
the interaction proteome. This information will provide
insights into the mechanisms of biological processes and
provide opportunities for controlling these processes by
interrupting key molecular interactions.
Surface plasmon resonance (SPR) is a method for characterizing macromolecular interactions. It is an optical
technique that uses the evanescent wave phenomenon to
measure changes in refractive index very close to a sensor
surface. The binding between an analyte in solution and
its ligand immobilized on the sensor surface results in a
change in the refractive index. The interaction is monitored in real time and the amount of bound ligand and
rates of association and dissociation can be measured with
high precision. Although SPR is a relatively new biophysical method, with the first commercial instrument being
introduced in 1990, its growth has been rapid. In the past
several years, SPR has taken its place as a mature biophysical technique for the analysis of molecular recognition
events. SPR biosensors have become standard instruments
in biochemical and biophysical research centers. There
have been yearly increases in the number of publications
in which SPR data is reported in an ever-wider variety of
In an age where fully sequenced genomes offer a wealth of
potential opportunities for insights into the molecular basis
of biological processes, our next great challenge is the
development of a complete set of proteomic interaction
maps. SPR can contribute to these efforts with rapid and
quantitative analysis of molecular interactions.
A number of commercial SPR biosensor instruments are
available [1]. Although BIAcore systems have dominated
the market since their introduction [2•], there are many
more competing SPR instruments now available, including
new systems introduced recently by Texas Instruments
and Aviv [3,4••]. A list of manufacturers and their web
addresses can be found in Table 1.
Since the introduction of their instruments, both BIAcore
and Affinity Sensors have marketed sensor chips with a
carboxymethylated dextran matrix and a streptavidinderivatized surface. Although thousands of publications
demonstrate the versatility and robustness of these surfaces, some systems are simply not compatible with these
immobilization strategies or sensor surface chemistries.
Consequently, several new commercial sensor chips have
been introduced in the past few years, permitting new
SPR-based applications. The new surface chemistries
include a carboxymethylated surface with a reduced
charge on the dextran matrix, several dextran-free ‘flat’
surfaces with different chemistries for immobilizing
ligands, a chelated nickel surface for binding His-tagged
ligands, and several hydrophobic surfaces designed to
allow assembly of lipid monolayers and analysis of
membrane systems [5,6].
SPR for studying biomolecular interactions:
insights into the mechanisms of molecular
recognition
Real strengths of the SPR biosensor technology are its
versatility and ease of use. It permits the analysis of
receptor–ligand interactions with a wide range of different
molecular weights, affinities and binding rates, and is compatible with a myriad of different chemical environments.
SPR is effective in studying interactions for a large range of
molecular weights of analytes. Experiments with analyte
masses ranging from hundreds of daltons [7] to whole-cell
binding [8] have been performed. Although the effective
affinity range of SPR has often been quoted to range from
nanomolar to micromolar, it is possible to extend this range
Surface plasmon resonance McDonnell
substantially, from sub-picomolar to greater than millimolar affinities [9]. The difficulties with measuring very high
affinity interactions are generally related to very slow
dissociation rates; for example, when koff = 10–6 s–1 it takes
approximately 104 seconds for 1% of bound material to
dissociate. Measuring these small signal changes is
experimentally challenging. High-affinity interactions can
instead be characterized using long sample injection times
and measuring equilibrium binding conditions over a range
of concentrations [9]. Care must be taken when measuring
binding interactions at very low affinities [10], when specific binding may be small compared with non-specific
binding to the sensor matrix or surface and instrument
noise and drift. Practical advice on improving data-collection methods, running proper controls and optimizing data
analysis methods has been reviewed recently [11••]. The
‘double referencing’ technique, a method for subtracting
reference data to remove small systematic signal deviations, is particularly helpful in the analysis of weak binding
interactions or low molecular weight analytes [11••].
SPR interaction analysis can be performed over a wide
range of chemical and environmental conditions (temperature, ionic strength, pH etc.). Analyzing binding kinetics
and thermodynamics over a range of different conditions
can give unique insights into binding reactions mechanisms. Andersson et al. [12] have described a systematic
approach for exploring buffer space, both as a means for
regenerating surface sensors and as a means for deriving
information about the binding event. Several groups have
measured binding affinities over a range of ionic strengths;
these studies describe the role of electrostatics for the
interaction [13,14]. The Debye–Huckel plot — a plot of
log(KA) versus log(ionic strength) — provides information
on the number of ions displaced during the binding event
[15]. One of the discriminating characteristics between
specific and non-specific protein–DNA binding is the
difference in their ionic strength dependence [16]. The
binding affinities of non-specific protein–DNA interactions are highly dependent on ionic strength, because of
the dominance of the electrostatic contributions mediated
by the negatively charged phosphates of the DNA backbone. Binding rates and affinities measured at different
temperatures also provide information on thermodynamic
parameters of the interaction. In addition to the well
known van’t Hoff plots (providing thermodynamic parameters ∆H, the change in enthalpy, and ∆S, the change in
entropy), the temperature dependence of the on- and
off-rates can give a direct value for the activation energies
(∆H‡,∆S‡,∆G‡) of these processes [17].
It has long been appreciated that protein folding events
can be described by complex energy landscapes and many
examples of intermediates along these folding pathways
have been characterized [18]. In contrast, the energy landscapes of molecular binding events are less well
characterized, although they are thought to be similar in
nature [19]. The ease of measuring the thermodynamic
573
Table 1
Manufacturers of SPR instruments.
SPR manufacturer
Internet address
System
BIAcore AB (Uppsala, Sweden)
http://www.biacore.com
Affinity Sensors (Franklin, MA)
http://www.affinity-sensors.com
Nippon Laser Electronics
(Hokkaido, Japan)
http://www.rikei.com
Artificial Sensing Instruments
(Zurich, Switzerland)
http://www.microvacuum.com
/products/biosensor
IBIS Technologies BV
(Enschede, The Netherlands)
http://www.ibis-spr.nl
Texas Instruments (Dallas, TX)
http://www.ti.com/spreeta
Aviv (Lakewood, NJ)
http://www.avivinst.com
BioTul AG (Munich, Germany)
http://www.biotul.com
Quantech Ltd (Eagan, MN)
http://www.quantechltd.com
BIAcore
IASys
SPR-670
OWLS
IBIS
TISPR
PWR-400
Kinomics
FasTraQ
parameters and the energy barriers for on- and off-rates by
SPR promises new insights into these processes. Using
kinetic and thermodynamic information, collected over a
range of temperatures and ionic strengths, Frisch et al.
[20••] have tentatively assigned a transition state for the
initial interaction between barnase and barstar. Several
groups have recently used SPR methods to characterize
the activation energies for both association and dissociation
events [21,22]. These kinds of studies will be critical
in understanding the energy landscapes that control
macromolecular interactions.
Analysis of kinetics and thermodynamics by SPR can be
used to understand the complex mechanisms of molecular
recognition events. Myszka et al. [23•] combined SPR with
isothermal titration calorimetry, analytical ultracentrifuge,
and structural information from X-ray crystallography to
describe the interaction between CD4 and gp120. Their data
suggest extensive structural rearrangements upon ligand
binding, which may have implications for HIV immune evasion and viral entry mechanisms. De Crescenzo et al. [24]
observed complex binding kinetics for the interaction
between TGF-α and the epidermal growth factor (EGF)
receptor. They considered several binding mechanisms and
found the biosensor data fit best to a conformational change
model. According to this model, ligand binding induces a
conformational change in the receptor resulting in receptor
dimerization. This mechanism is consistent with other biophysical studies of EGF receptor–ligand interactions and is
thought be important for EGF receptor-mediated cellular
activation. Gunnarsson et al. [25] used SPR to study conformation variants of human α2-macroglobulin, identifying a
574
Analytical techniques
Figure 1
(a)
(b)
(c)
Association
Association
Dissociation
100
Dissociation
100
80
80
60
60
RU
RU
40
40
20
20
0
0
0
200
400
0
600
200
Time (s)
400
600
Time (s)
(d)
(e)
100
100
90
90
80
80
70
70
RU
RU
60
60
50
50
40
40
0
100
200
300
Time (s)
400
0
100
200
300
400
Time (s)
Current Opinion in Chemical Biology
Insights into molecular recognition processes using SPR. (a) In the
interaction between IgE and FcεRIα, Trp87 makes important energetic
contributions to binding. BIAcore SPR analysis of the Cε3-4 domains
from IgE binding to (b) FcεRIα wild type and (c) a Trp87Asp mutant
demonstrate the effect of this mutation on interaction kinetics. Wild type
(red circles) and mutant (blue squares) dissociation curves are fit (black
line) to (d) a single-component exponential decay or (e) a two-component
system. The IgE–FcεRI interaction shows biphasic kinetics [26], but while
the second component makes only a minor contribution to the wild-type
interaction, this biphasicity is markedly exaggerated by the Trp87Asp
mutation. The SPR data demonstrate the effect of this mutation on the
free energy (∆G) of binding, and also offer insights into the complex
energy landscape of this binding event, describing the pathways used in
the transition from free to bound states. RU, resonance units.
site involved in exposure of a ligand recognition site.
Deviations from ideal 1:1 Langmuir models have also been
observed in a large number of other systems and often this
information has been used to propose mechanisms for
ligand-mediated activation events [26,27] (see Figure 1).
Several examples have been identified in which interaction
rates are more descriptive of a given biological process than
the equilibrium binding affinities. For example, Leferink
et al. [28] have described growth factor interactions with
ErbB-1 in which the dynamic rate constants correlate better
with mitogenic activity than do the equilibrium constants.
McDonnell et al. [29] identified a role for the Cε2 domain
from immunoglobulin E (IgE) in allergic responses. Removal
of this domain from IgE has a small effect on the overall
affinity of IgE for its receptor FcεRI but has a marked effect
on the off-rate. Cε2 thus contributes to the unusually slow
off-rate of IgE, which is an important factor in IgE-mediated
mast cell sensitization as part of the allergic response.
The analysis of complex kinetics has been aided by the
introduction of improved data-fitting software. In addition
to manufacturer-supplied curve-fitting software, several
excellent freeware programs are also available including
CLAMP (http://www.cores.utah.edu/interaction/clamp.htm)
[30] and SPRevolution (http://www.bri.nrc.ca/csrg/equip.
htm) [24]. Using sensible experimental design and data
collection methods [31], and applying the improved data
analysis, including modelling mass transport effects [32],
one can essentially eliminate experimental artefacts, which
were common in early SPR studies [33,34], and allow
Surface plasmon resonance McDonnell
confidence in interpreting kinetic data into insights into
biological mechanisms.
SPR in membrane studies
Before SPR gained popularity as a technique to analyze
biomolecular interactions, plasmon resonance spectroscopy was used for many years by material scientists for
measurements of surface and optical properties of molecular films and interfaces [35,36]. It seems only natural, then,
that SPR could be a powerful approach for studying
biological membrane events. The recent introduction
of sensor surfaces specifically designed to allow study of
membrane interactions has resulted in a large number of
new studies of membrane-associated systems by SPR. The
planar nature of the sensor surface and the ability to specifically orient immobilized ligands may make SPR a better
membrane surrogate than traditional solution studies. Erb
et al. [37] have characterized one of the new BIAcore
sensor chips (L1) designed for liposome absorption. Using
atomic force microscopy and fluorescence microscopy, they
have demonstrated that lipids form a homogeneous monolayer on the surface of the chip, suggesting a successful
membrane mimic. Liposome-covered sensor surfaces have
been used in an assay for lipid absorption for a panel of 27
drugs and showed a strong correlation with passive intestinal absorption [38]. Celia et al. [39•] established a lipid
monolayer using a chelated-nickel lipid and immobilized
and oriented a major histocompatibility complex (MHC)
molecule through a His-tag tail, and then measured binding events between the T cell receptor and the oriented
MHC molecule. X-ray diffraction studies of the twodimensional crystals of the monolayer-bound MHC
molecules established that the protein had maintained the
desired orientation. Several recent reports describe the
characterization of membrane-integrated G-protein-coupled receptors [40–42]. Using a variation of SPR on a
home-built instrument (the predecessor of the commercial
system from Aviv) Salamon et al. [4••] used coupled
plasmon-waveguide resonance (CPWR) spectroscopy to
study interactions and conformational change in the
rhodopsin–transducin system. The CPWR spectrum has a
higher information content than the traditional SPR
measurement, which typically records only a change in the
resonance angle. CPWR may have some important
advantages in the study of anisotropic membrane systems.
SPR in proteomics and drug discovery
The general versatility of SPR methods, the ease of automation, and the lack of labeling requirements make it a promising
tool for large-scale screening for binding events, both for small
molecules in drug discovery efforts or for macromolecules in
large-scale ligand fishing experiments. The improved
sensitivity of new biosensor instruments routinely permits the
detection of small molecular weight (<500 Da) analytes, even
for low-affinity interactions (KD > 1 mM). Adamczyk et al. [43]
used SPR as an immunoassay to monitor binding of thyroxin
analogs to an immobilized monoclonal antibody. The La Jolla
Pharmaceutical Company has used SPR in the clinical
575
development of a new drug for the treatment of the autoimmune lupus [44]. A review by Myszka and Rich [45] discusses
recent progress in SPR in drug discovery efforts. This report
also describes a prototype microarray chip, a sensor surface
with 64 individual immobilization sites in a single flow cell
(the standard BIAcore chip has four independent flow cells).
SPR-based arrays offer the opportunity to move towards larger-scale matrices of receptor–ligand interactions, the type of
analysis that will be required to build complete proteomeinteraction maps. A great number of different protein biochip
technologies are being pursued [46•,47–49]. One promising
approach is the combination of protein-chip-based technologies with mass spectrometry (MS) (see for example [50]).
Several groups have integrated SPR and MS for affinity-based
capture and characterization of ligands [51,52]. Nelson et al.
[53] discuss performing matrix-assisted laser desorption/ionisation (MALDI)-MS directly on ligand-bound biosensor
surfaces. A fully in-line system combining an SPR biosensor
and electrospray tandem MS has been described [54•]. The
SPR platform allows the detection, capture and subsequent
digestion and delivery of nanomolar to femtomolar levels of
ligand for MS analysis. SPR/MS is a rapid and powerful
approach for identifying binding partners from complex
mixtures of components. Combining this technology with
larger microarrays makes it a feasible approach for large-scale
ligand-fishing experiments or interaction proteomics analyses.
Conclusions
With the introduction of a number of new SPR instruments
and a series of novel sensor surfaces and chemistries, the
impact of SPR biosensors on molecular interaction studies
will continue to grow. The ability to form stable membrane
surfaces on biosensor chips will greatly simplify binding
analyses in membrane systems, making this important class of
biological systems far more accessible to quantitative analysis.
With improved experimental design and data analysis
methods, it is now easier to obtain high-quality data for the
kinetic and thermodynamic parameters of intermolecular
interactions. These data promise additional insights into the
mechanisms of molecular binding events, which will be
important in rational drug design of inhibitors of macromolecular interactions. SPR and other protein-chip-based
technologies are beginning to show promise in larger-scale
interaction studies, both for small-molecule analysis and
macromolecular ligand-fishing experiments. Great potential
exists for interfacing SPR and MS in a broader microarray
approach for characterizing proteome-wide interaction maps.
Acknowledgements
I acknowledge support from the EP Abraham Fund. I am grateful to
E Garman, L Hewitt, R Lewis and M Noble for critical review of the
manuscript and H Gould and B Sutton for helpful discussions.
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•
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J Biol Inorg Chem (2005) 10: 874–886
DOI 10.1007/s00775-005-0037-x
O R I GI N A L A R T IC L E
Hesheng Ou Æ Limei Yan Æ Devkumar Mustafi
Marvin W. Makinen Æ Matthew J. Brady
The vanadyl (VO2+) chelate bis(acetylacetonato)oxovanadium(IV)
potentiates tyrosine phosphorylation of the insulin receptor
Received: 10 July 2005 / Accepted: 20 September 2005 / Published online: 19 October 2005
SBIC 2005
Abstract We have compared the insulin-like activity of
bis(acetylacetonato)oxovanadium(IV) [VO(acac)2], bis
(maltolato)oxovanadium(IV) [VO(malto)2], and bis(1-Noxide-pyridine-2-thiolato)oxovanadium(IV) [VO(OPT)2] in
differentiated 3T3-L1 adipocytes. The insulin-like
influence of VO(malto)2 and VO(OPT)2 was decreased
compared with that of VO(acac)2. Also, serum albumin
enhanced the insulin-like activity of all three chelates
more than serum transferrin. Each of the three VO2+
chelates increased the tyrosine phosphorylation of
proteins in response to insulin, including the b-subunit
of the insulin receptor (IRb) and the insulin receptor
substrate-1 (IRS1). However, VO(acac)2 exhibited the
greatest synergism with insulin and was therefore
further investigated. Treatment of 3T3-L1 adipocytes
with 0.25 mM VO(acac)2 in the presence of 0.25 mM
serum albumin synergistically increased glycogen
accumulation stimulated by 0.1 and 1 nM insulin, and
increased the phosphorylation of IRb, IRS1, protein
kinase B, and glycogen synthase kinase-3b. Wortmannin suppressed all of these classical insulin-signaling
activities exerted by VO(acac)2 or insulin, except for
tyrosine phosphorylation of IRb and IRS1. Additionally, VO(acac)2 enhanced insulin signaling and
metabolic action in insulin-resistant 3T3-L1 adipocytes.
Cumulatively, these results provide evidence that
VO(acac)2 exerts its insulin-enhancing properties by
directly potentiating the tyrosine phosphorylation of
the insulin receptor, resulting in the initiation of insulin
metabolic signaling cascades in 3T3-L1 adipocytes.
H. Ou Æ L. Yan Æ M. J. Brady (&)
Department of Medicine and Committee on Molecular Metabolism
and Nutrition, The University of Chicago, MC1027, 5841 S.
Maryland Ave, Chicago, IL 60637, USA
E-mail: [email protected]
Tel.: +1-773-7022346
Fax: +1-773-8340486
D. Mustafi Æ M. W. Makinen
Department of Biochemistry and Molecular Biology,
The University of Chicago, Chicago, IL 60637, USA
Keywords Adipocyte Æ Diabetes Æ Insulin receptor and
insulin receptor substrate Æ Insulin signaling Æ Tyrosine
phosphorylation Æ Vanadyl
Abbreviations BSA: Bovine serum albumin Æ DMSO:
Dimethyl sulfoxide Æ ENDOR: Electron–nuclear
double resonance Æ EPR: Electron paramagnetic
resonance Æ GSK: Glycogen synthase kinase Æ HEPES:
N-(2-Hydroxyethyl])piperazine-N¢-(2-ethanesulfonic
acid), sodium salt Æ IRb: b-subunit of the insulin
receptor Æ IRS1: Insulin receptor substrate-1 Æ KRBH:
Kreb’s Ringer’s buffer Æ PBS: Phosphate-buffered
isotonic saline Æ PI: Phosphatidyl inositol Æ PKB:
Protein kinase B Æ VO(acac)2: Bis(acetylacetonato)
oxovanadium(IV) Æ VO(malto)2: Bis(maltolato)
oxovanadium(IV) Æ VO(OPT)2: Bis(1-N-oxide-pyridine2-thiolato)oxovanadium(IV)
Introduction
Studies have now convincingly demonstrated blood glucose lowering effects in diabetic humans [1–4] and laboratory animals [5–7] and enhanced lipogenesis [8, 9] and
glucose uptake [10] in whole cells by vanadium compounds. Vanadium, occurring between Ca2+ and Zn2+ in
the first transition-metal series, exhibits complex chemistry because of multiple oxidation states [11–13]. This
factor has led to confusion in identifying the antidiabetic
agent, for vanadium compounds of oxidation states IV
and V are both associated with insulin-enhancing activity—but through different mechanisms. Furthermore,
vanadyl (VO2+) compounds, of oxidation state IV, under
physiological conditions are subject to oxidation by a
variety of oxidants, including molecular oxygen [14]; and
vanadate compounds, of oxidation state V are thought to
undergo reduction to state IV in the cell [14–16]. For these
reasons, the mechanisms by which vanadium compounds
mediate antidiabetic effects in vivo are poorly described
and incompletely understood.
875
The chemical bonding structures of the three organic
chelates of VO2+ that have received the greatest
attention through laboratory studies are illustrated in
Fig. 1. These VO2+ chelates exhibit significantly enhanced insulin-mimetic activity in diabetic laboratory
animals or adipocyte cells over that of inorganic VO2+
introduced as VOSO4 [5–10]. Although these observations suggest that the increase is at least in part due to
the increased lipophilic character of the chelate, they
also indicate that the structure of the organic chelating
ligand and its electronic structure, i.e., bonding interactions with the VO2+ moiety, are important factors
governing the reactivity of VO2+ chelates with biological macromolecules.
Organic chelates of VO2+, of which the bis(maltolato)oxovanadium(IV) [VO(malto)2] compound is presently the most widely studied example in the literature
[17–22], offer the most likely vehicle for drug design
because the structural properties of the organic ligands
chelating the central V4+ ion could be synthetically
molded to increase specificity of action. Since inorganic
VO2+, existing as the penta-aquo vanadyl cation
[VO(H2O)5]2+ in solution [23, 24], is itself associated
with insulin-like activity [25, 26], an important property
of the chelating ligand is its intrinsic binding affinity with
which the central VO2+ moiety is retained in the complex, particularly in the presence of biological macromolecules such as serum transport proteins. These
aspects have not received uniform attention, and the
insulin-like activity of a variety of VO2+ chelates has
been measured in whole animal and cellular systems
with little regard to the relative proportions of intact
chelate, inorganic VO2+, or partial, hemichelated species into which the compound may have dissociated.
Since the insulin-mimetic properties of each chemical
species are likely to differ according to specific activity
and structural basis and a variety of cellular, enzyme,
and whole animal assay methods for measuring antidiabetic activity are employed, it is often not evident which
chemical species of vanadium is responsible for the observed effects.
We have argued that the organic chelating ligands of
the VO2+ compounds illustrated in Fig. 1 are likely to
facilitate binding to proteins in the blood stream [10].
Binding to serum albumin, for instance, as the
major serum transport protein, may stabilize VO2+
against oxidation or result in formation of a specific
[protein/VO2+ chelate] adduct that is recognized at the
membrane surface of target cells. In support of these
ideas, we have observed that bis(acetylacetonato)oxovanadium(IV) [VO(acac)2] (Fig. 1a) forms an adduct
of 1:1 stoichiometry with serum albumin and that
its insulin-mimetic effect, measured as the uptake of
2-deoxy-D-[1-14C]glucose by differentiated, cultured
3T3-L1 adipocytes, is greatly enhanced in the presence
of albumin [10]. To investigate the structural origins of
the insulin-enhancing activity of VO2+ chelates further,
we compared the stability and structures in solution of
the three organic chelates of VO2+ illustrated in Fig. 1,
the influence of albumin and transferrin on their insulin-enhancing activity in cultured 3T3-L1 adipocytes,
and their interactions with components of the insulinsignaling pathway. Our results lead to the conclusion
that VO(acac)2 most strongly promotes the autophosphorylation and tyrosine kinase activity of the insulin
receptor, enhancing the phosphorylation of tyrosine
residues on insulin receptor substrate-1 (IRS1) in a
dose-dependent manner that is synergistic with added
insulin. Since our observations run counter to the
conclusions of the group of Shechter [8, 9, 27] that the
influence of inorganic VO2+ and of VO2+ chelates lies
downstream from the insulin receptor involving a
cytosolic protein tyrosine kinase, it is important to
resolve the origin of these apparently contradictory
results and to identify target enzymes of VO2+ chelates
to assign the molecular basis of their insulin-enhancing
activity.
Fig. 1 Comparison of the chemical bonding structures of organic
chelates of VO2+ with insulin-enhancing activity. a Bis(acetylacetonato)oxovanadium(IV) [VO(acac)2], b bis(maltolato)
oxovanadium(IV) [VO(malto)2], c bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV) [VO(OPT)2]
Materials and methods
Materials
Cell culture reagents and calf serum were supplied by
Mediatech (Herndon, VA, USA). Fetal bovine serum
was obtained from Hyclone (Logan, UT, USA). Porcine
insulin and all other chemicals were from Sigma (St.
Louis, MO, USA). D-[U-14C]glucose (317 mCi/mmol)
876
was obtained from MP Biomedicals (Irvine, CA, USA),
while UDP-[U-3H]glucose (60 Ci/mmol) was supplied
by American Radiolabeled Chemicals (St. Louis).
Enhanced chemiluminescence reagents and Protein A
Sepharose beads were purchased from Amersham Biosciences (Piscataway, NJ, USA), and GF/A filters were
supplied by Whatman (Haverhill, MA, USA). Commercial sources of antibodies were as follows:
anti-phospho-GSK-3b (Ser9), where GSK-b is glycogen
synthase kinase-3b, and anti-phospho-PKB (Thr308),
where PKB is protein kinase B, were purchased from
Cell Signaling Technology (Beverly, MA, USA), antiphosphotyrosine (clone 4G10) was supplied by Upstate
Cell Signaling Solutions (Lake Placid, NY, USA), antiinsulin receptor b-subunit (C-19) was from Santa Cruz
Biotechnology (Santa Cruz, CA, USA), and anti-IRS1
polyclonal was generated by injection of rabbits with a
GST-fusion construct comprising the N-terminus of
mouse IRS1. Horseradish peroxidase-conjugated goat
antirabbit and goat antimouse immunoglobulin G were
obtained from Bio-Rad (Hercules, CA, USA).
Vanadyl sulfate hydrate and VO(acac)2 were purchased
from Sigma-Aldrich (Milwaukee, WI, USA). 1-N-Oxide2-thiolato-pyridine (99%) was obtained from Research
Chemicals (Heysham, Lancashire, UK) and used without
further purification for preparation of crystalline bis(1-Noxide-pyridine-2-thiolato)oxovanadium(IV) [VO(OPT)2]
according to the procedure of Sakurai et al. [7]. Crystalline
VO(malto)2 was a gift from C. Orvig and K. H. Thompson of the University of British Columbia. Fatty acid free
bovine serum albumin (BSA) and bovine apotransferrin
(98%) were from Sigma. All other reagents were as previously described [10].
Cell culture
3T3-L1 fibroblasts were maintained and differentiated
into adipocytes, as previously reported [10, 28]. Cells
were used 5–10 days after completion of the differentiation protocol, when more than 95% of the cells contained lipid droplets. Prior to experiments, the cells were
washed twice with Kreb’s Ringer’s buffer (KRBH)/0.5%
BSA/5 mM glucose and incubated in the same medium
for 3 h. The cells were then washed twice with KRBH
lacking BSA, and then placed in KRBH containing a
VO2+ chelate in 1:1 molar ratio with BSA in the absence
or presence of the indicated concentration of insulin.
After treatment of adipocytes under these conditions for
5–15 min, the cells were used for glycogen synthesis
assay, or placed onto ice and washed three times with
Phosphate-buffered isotonic saline (PBS).
Preparation of cell lysates
After washing with PBS, the cells were scraped directly
into homogenization buffer [50 mM (N-(2-hydroxyethyl)
piperazine-N¢-(2-ethanesulfonic acid), sodium salt
(HEPES), pH 7.5, 150 mM NaCl, 10% glycerol, 0.5%
Triton X-100, 2 mM EDTA, 10 mM NaF, with the
protease inhibitors aprotinin (10 lg/ml) and benzamidine (10 lM) added just before use] for immunoblotting
experiments. Lysates were centrifuged for 10 min at
10,000g at 4 C, and the supernatants were transferred
to new microfuge tubes. Immunoblotting was performed
as previously described [29].
Immunoprecipitations
Cell lysates were prepared as just described, and precleared for 15 min with empty Protein A Sepharose
beads to remove any nonspecific protein binding to the
immunocomplex. A 10-ll aliquot of the indicated
antiserum was added to the supernatant, and the sample
was mixed for 1 h at 4 C. A 50-ll aliquot of a 50%
protein A sepharose bead slurry was added, and the
samples were mixed for 30 min. The sample was centrifuged for 2 min at 800g and 4 C, and the pelleted
antibody–bead complex was then washed three times
with 1 ml of homogenization buffer. Bound protein
was eluted from the beads using 80 ll of 1X Laemmli
sample buffer and boiling for 2 min. Samples were separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and analyzed by anti-phosphotyrosine immunoblotting [29].
Metabolic assays
Glycogen synthesis assays were performed as previously
reported [28, 30]. Briefly, after serum starvation, 3T3-L1
adipocytes in 12-well dishes were incubated as described
earlier in the presence of the indicated vanadyl compound with or without insulin for an additional 15 min.
Subsequently, the cells were incubated with 5 mM
14
D-[U- C]glucose (2 lCi per well) for 45 min at 37 C.
The cells were then washed three times with ice-cold PBS
and solubilized in 30% (w/v) KOH. Cellular glycogen
was precipitated with 70% ethanol and radiolabeled
glucose incorporation into glycogen was determined by
liquid scintillation counting.
Glycogen synthase activity was determined as
described previously [28] with some modifications.
Cells in 12-well dishes were serum-deprived, then
incubated with the indicated vanadyl compound with
or without insulin for 15 min at 37 C. After washing
with ice-cold PBS three times, the cells were scraped
into 500 ll of glycogen synthase assay buffer [50 mM
tris(hydroxymethyl)aminomethane)–HCl, pH 7.8, 10 mM
EDTA, 100 mM NaF, 0.5% Triton X-100] and centrifuged (10,000g for 10 min). To measure glycogen
synthase activity, a 50-ll aliquot of the supernatant
(50–100 lg protein) was added to an equal volume
of buffer lacking detergent, containing 10 mM UDP[U-3H]glucose (0.1 lCi/lmol) and 16 mg/ml glycogen,
in the presence or absence of 20 mM glucose-6-phosphate. After 15-min incubation at 37 C, the assay
877
tubes were chilled for 15 min in an ice bath. A 90-ll
aliquot of the reaction mixture was then spotted on
2.4-cm GF/A Whatman filter disks, and glycogen was
precipitated by immersion in 70% ethanol at 4 C. Free
UDP-[U-3H]glucose was removed by washing the filters
three times for 10 min each time in 70% ethanol, the
last two washings occurring at room temperature. The
filters were air-dried, and incorporation of radioactivity
was determined by liquid scintillation counting.
Electron paramagnetic resonance and electron–nuclear
double resonance
For comparative studies of the stability of VO2+ chelates,
stock solutions were prepared by dissolving the crystalline compound in a small volume of dimethyl sulfoxide
(DMSO) followed by dilution to the desired concentration with N2-purged KRBH buffered with 10 mM
HEPES to pH 7.4. The final solutions contained no more
than 10% (v/v) DMSO. No evidence of chelate displacement by DMSO was detectable by electron paramagnetic resonance (EPR) or electron–nuclear double
resonance (ENDOR). Also, no evidence of binding of
DMSO to inorganic VO2+ in solution or to the axial
coordination site in VO2+ chelates was found on the basis
of ENDOR spectra. Aliquots of these solutions were removed under a N2 atmosphere as a function of time and
frozen immediately and kept in liquid N2 until used for
collection of EPR and ENDOR spectra.
EPR and ENDOR spectra were recorded with an Xband Bruker ESP 300E spectrometer equipped with a
cylindrical TM110 ENDOR cavity and an Oxford
Instruments ESR910 liquid helium crysotat and Bruker
ENDOR digital accessories, as previously described [31,
32]. The spectrometer was equipped with a complete
computer interface (Bruker ESP3220 data system) for
spectrometer control and data acquisition and processing. Typical experimental conditions for ENDOR
measurements were as follows: temperature 20 K,
microwave frequency 9.45 GHz, incident microwave
power 64 lW (full power 640 mW at 0 dB), rf power
50–70 W, rf modulation frequency 12.5 kHz, and rf
modulation depth 10–20 kHz. The static laboratory
magnetic field was not modulated for ENDOR.
Results
Comparative insulin-like activity of VO2+ chelates
In efforts to identify the VO2+ species that potentiate
insulin signaling in 3T3-L1 adipocytes, we compared
the patterns of tyrosine phosphorylated proteins stimulated by VO(acac)2, VO(malto)2, and VO(OPT)2, in
the presence and absence of insulin. As seen in Fig. 2,
treatment of cells with each of the VO2+ chelates in
the presence or absence of insulin resulted in an array
of tyrosine phosphorylated proteins. However, it is
evident that the intensity of only two bands migrating
at 90 and 180 kDa increased in a dose-dependent
manner with addition of insulin. These proteins were
presumed to be the b-subunit of the insulin receptor
(IRb) and IRS1, respectively, and this supposition was
subsequently confirmed (see later). In Fig. 2a, it is
evident that the intensity of tyrosine phosphorylation,
particularly of the insulin-sensitive proteins at 90 and
180 kDa, was significantly greater with VO(acac)2 than
with VO(malto)2 or VO(OPT)2.
In earlier studies, the group of Shechter [9, 27] concluded that the insulin-mimetic action of VO2+ and
VO(acac)2 involves only activation of a postreceptor
pathway. To confirm that VO(acac)2 treatment of 3T3L1 adipocytes increased tyrosine phosphorylation of
IRS1 and IRb, immunoprecipitations were performed,
using a specific antibody for each protein. 3T3-L1
adipocytes were treated in the presence or absence of
10 nM insulin with and without 0.25 mM VO(acac)2 for
5 min, lysates were prepared and subjected to anti-IRS1
or anti-IRb immunoprecipitation, and samples were
then analyzed by anti-phosphotyrosine immunoblotting.
Insulin increased the tyrosine phosphorylation of the
180-kDa IRS1 protein and the 90-kDa IRb subunit
(Fig. 2b, c), confirming the identity of these proteins in
the anti-phosphotyrosine immunoblot shown in Fig. 2a.
These results demonstrated that VO(acac)2 increased the
tyrosine phosphorylation of the insulin receptor and its
immediate downstream target IRS1 in 3T3-L1 adipocytes.
In Fig. 3, we compare the influence of serum albumin and serum transferrin on the insulin-mimetic
response of each VO2+ compound. While the intensity
of the immunoblots was uniformly greater for each
VO2+ chelate in the presence of serum albumin than in
the presence of transferrin, the pattern of tyrosine
phosphorylated proteins remained the same. However,
close inspection of the immunoblots showed that the
ordering of intensities was dependent on the protein
added to the assay mixture. In the presence of albumin,
the relative intensities followed the order: VO(acac)2
VO(OPT)2>VO(malto)2@VOSO4>control. On the
other hand, in the presence of transferrin, the ordering
of intensities was VO(acac)2VO(OPT)2>VO(malto)2@VOSO4@control, with virtually no differentiating
feature among the last three systems. Furthermore, the
intensity of the immunoblots was responsive to
increasing concentrations of insulin only for VO(acac)2
and VO(OPT)2 in the presence of serum transferrin,
while the intensity of the immunoblots elicited by
VOSO4 and VO(malto)2 remained unaltered and did
not differ from that of the control. These observations
indicated that the intensities of the immunoblots for
each VO2+ chelate were sensitive to insulin only in the
presence of albumin.
Figure 4 compares the intensity of tyrosine phosphorylation of signaling proteins promoted by VO
(acac)2 in the presence and absence of 10 nM insulin. In
the absence of insulin, lanes 1–6 showed that there is a
878
Fig. 2 Comparison of effects of
vanadyl chelates on tyrosine
phosphorylation in 3T3-L1
adipocytes. Cells were serumstarved for 3 h, and then
incubated in Kreb’s Ringer’s
buffer (KRBH) + 0.25 mM
bovine serum albumin, with the
indicated concentration of
insulin, in the presence of
0.25 mM VO(acac)2,
VO(malto)2, or VO(OPT)2 for
5 min. a Cellular lysates
separated by sodium dodecyl
sulfate polyacrylamide gel
electrophoresis and transferred
to nitrocellulose for
immunoblotting with antiphosphotyrosine antibody.
Alternatively, lysates were
subjected to
immunoprecipitation using
anti-insulin receptor substrate-1
(anti-IRS1) (b) or the b-subunit
of the anti-insulin receptor
(anti-IRb) (c) antibodies, and
samples were then analyzed by
anti-phosphotyrosine
immunoblotting. All results are
representative of two to four
independent experiments
monotonic increase in the intensity of IRb with
increasing concentration of VO(acac)2 in the incubation
medium. In the presence of 10 nM insulin, as shown
through lanes 7–12, there was similarly a monotonic
increase in the intensity of IRb with increasing concentrations of VO(acac)2. However, in the presence of
insulin the bands belonging to IRb were more intense
than in the absence of insulin. These observations indicated that VO(acac)2 acts synergistically with insulin,
increasing the tyrosine phosphorylation of the insulin
receptor potentiating the same enzymatically controlled
pathway.
Comparative spectroscopic studies of the relative
stability of VO2+ chelates in solution
We presumed that the insulin-enhancing action of
organic chelates of VO2+ was likely to be a function of
their relative chemical stability regulated by the binding
affinity of the organic ligand for VO2+ and the tendency
of the VO2+ chelate to be oxidized. Therefore, we examined the stability of the organic VO2+ chelates in KRBH
medium used for incubation of adipocytes in metabolic
assays. To determine the chemical stability with time, we
selected a combined EPR and ENDOR approach to
monitor the presence of intact VO2+ chelate. Figure 5
illustrates progress curves showing a time-dependent
decrease in the intensity of EPR and ENDOR absorptions of VO(acac)2, VO(malto)2, and VO(OPT)2 in
KRBH medium. Analysis showed that the data corresponded to a single-exponential decay with half-lives
(t1/2) of (4, 6, and 4,000 days or more for VO(malto)2,
VO(OPT)2, and VO(acac)2, respectively. Since the EPR
intensity monitors the concentration of the paramagnetic VO2+ ion while the ENDOR intensity is specific for the content of covalent hydrogens of ligands
attached to the paramagnetic VO2+ ion, the parallel
879
Fig. 3 Comparison of effects of
vanadyl chelates on tyrosine
phosphorylation in 3T3-L1
adipocytes in the presence of
serum albumin or transferrin.
Insulin in indicated amounts
and 0.25 mM VO2+ chelates
were added in the presence of
either 0.25 mM serum albumin
or 0.25 mM serum transferrin.
Arrows point to IRS1 and IRb
components identified by
specific antibody reaction. It is
evident that the intensity of
tyrosine phosphorylated
proteins upon addition of
0.25 mM VOSO4 in the
presence or absence of insulin
did not differ discernably from
basal conditions
decrease in intensity reflects loss of intact VO2+ chelate.
The results in Fig. 5, thus, demonstrate that the stability
of VO(acac)2 in solution as an intact complex far
exceeded that of the other two VO2+ chelates under
conditions comparable to those employed for measuring
glucose uptake by adipocytes. Other investigators have
also observed diminution of the EPR absorption of
VO2+ chelates as a function of time comparable to the
Fig. 4 Dose response of VO(acac)2-stimulated tyrosine phosphorylation in the presence and absence of insulin. 3T3-L1 adipocytes were
serum-starved for 2.5 h prior to addition of 10 nM insulin to half of
the wells, and varying concentrations of VO(acac)2. After 5 min, cellular lysates were prepared and analyzed by anti-phosphotyrosine
immunoblotting. Concentrations of VO(acac)2 used: lanes 1 and 7
0 mM; lanes 2 and 8 0.01 mM; lanes 3 and 9 0.05 mM; lanes 4 and
10 0.1 mM; lanes 5 and 11 0.25 mM; and lanes 6 and 12 0.5 mM.
The immunoblot shown is representative of three independent
experiments
880
Fig. 5 Comparison of the time-dependent decrease in intensity of
electron paramagnetic resonance (EPR) absorption and in the
intensity of electron–nuclear double resonance (ENDOR) absorptions of covalent hydrogens specific for the ligand of VO2+ chelates
in KRBH. Aliquots of solutions of VO(acac)2, VO(malto)2, and
VO(OPT)2, prepared as described in the ‘‘Materials and methods’’
and kept at ambient temperature (22 C), were removed at the
indicated times under N2 with a Hamilton air-tight syringe
equipped with a Teflon catheter, transferred directly into EPR
sample tubes, and immediately frozen and stored in liquid N2. EPR
and ENDOR spectra were collected at (0 K as previously described
[28, 29]. The peak-to-peak amplitude of the 3/2^ component of
the EPR absorption spectrum and of the A^ features of the
ENDOR spectrum with H0 at the 3/2^ setting [10, 18] are plotted
as IEPR and IENDOR, respectively, normalized to the corresponding
amplitudes at time zero. The data were found to adhere to firstorder exponential decay with use of the program Origin (Microcal
Software, Northampton, MA, USA), from which the half-lives
were estimated as 4, 6, and 4,000 days or more for VO(malto)2,
VO(OPT)2, and VO(acac)2, respectively
results described here for VO(malto)2 and VO(OPT)2
[33]. Since the insulin-enhancing activity of VO(acac)2
far exceeded that of the other two chelates and was
synergistic with added insulin, as shown through Figs. 2
and 4, we used only VO(acac)2 in further investigations
of its influence on other components of the insulin-signaling pathway in 3T3-L1 adipocytes in view of its
superior chemical stability.
effect under all conditions tested (Fig. 6a). Figure 6b
shows that the intensity of immunoblots indicative of
phosphorylation of GSK-3b in the absence of insulin
(lanes 1–6) is dependent on the concentration of VO
(acac)2 in the incubation medium. In the presence of
10 nM insulin (lanes 7–12), there is also an increase in
the intensity of immunoblots dependent on the concentration of VO(acac)2; however, the influence of increasing concentrations of VO(acac)2 in the presence of
10 nM insulin quickly saturates the response and synergistic activity is not observed as readily as in Fig. 6a.
Cumulatively, these observations demonstrate that the
insulin-mimetic action of VO(acac)2 potentiating the
phosphorylation of PKB and GSK-3b is synergistic with
insulin and is not replicated by VOSO4.
Influence of VO(acac)2 on phosphorylation
of insulin-signaling proteins
In our earlier investigation of the insulin-like action of
organic chelates of VO2+, we demonstrated that
VO(acac)2 is superior to other VO2+ chelates enhancing
glucose uptake in 3T3-L1 adipocytes [10]. In Fig. 2, it is
seen that VO(acac)2 increased the intensity of tyrosine
phosphorylation of IRS1 in the absence and presence of
10 nM insulin. Therefore, to examine the downstream
effects of enhanced IRS1 phosphorylation, we determined the phosphorylation states of PKB and GSK-3b
by immunoblotting. In Fig. 6a, it is seen that immunoblotting indicative of serine/threonine phosphorylation
of PKB and GSK-3b was significantly more intense in
the presence of VO(acac)2 for all levels of added insulin.
In contrast, inclusion of VOSO4 had little discernable
Potentiation of glycogen synthesis and glycogen
synthase activation by VO(acac)2
A primary metabolic effect of insulin in adipocytes
and muscle is to increase incorporation of cellular
glucose into glycogen through the coordinated translocation of GLUT4 and the activation of glycogen
synthase. As shown in Fig. 7, the influence of VO
(acac)2 on glucose incorporation into glycogen in the
absence of insulin was significantly greater than the
881
Fig. 6 Influence of VO(acac)2 on the phosphorylation states of
protein kinase B (PKB) and glycogen synthase kinase-3b (GSK-3b).
a 3T3-L1 adipocytes were serum-starved for 3 h, and treated with
the indicated concentration of insulin, in the presence of 0.25 mM
VO(acac)2 or VOSO4 for 5 min. Lysates were prepared and the
phosphorylation states of PKB and GSK-3b were determined by
immunoblotting with phospho-specific antibodies. Bands corresponding to phospho-PKB and phospho-GSK-3b (pGSK-3b) are
indicated by arrows. b Serum-starved 3T3-L1 adipocytes were
stimulated for 5 min with varying amounts of VO(acac)2, in the
absence (basal) or presence of 10 nM insulin. Lysates were
prepared and analyzed by immunoblotting using anti-pGSK-3b
antibody. Concentrations of VO(acac)2 used: lanes 1 and 7 0 mM;
lanes 2 and 8 0.01 mM; lanes 3 and 9 0.05 mM; lanes 4 and 10
0.1 mM; lanes 5 and 11 0.25 mM; and lanes 6 and 12 0.5 mM. All
results are representative of three independent experiments
basal rate and also greater than that for VOSO4.
Furthermore, the action of VO(acac)2 was synergistic
with submaximal insulin in promoting incorporation
of glucose into glycogen (Fig. 7a). In the presence of
10 nM insulin, the capacity of the adipocytes for glycogen synthesis was saturated, and the contribution of
the chelate or of VOSO4 above the influence of the
hormone was no longer detectable from that observed
at lower insulin concentrations.
Since glycogen synthase activity is normally stimulated by insulin, we examined whether the influence of
the VO2+ chelate in augmenting glycogen synthesis
derives from changes in activity of this rate-limiting
enzyme. As illustrated in Fig. 7b, the glycogen synthase activity ratio was increased with VO(acac)2 in
the incubation medium both in the absence and in the
presence of insulin. However, this result does not
distinguish between VO(acac)2 acting upstream of
glycogen synthase vs. direct activation of glycogen
synthase.
To address this issue, 3T3-L1 adipocytes were
pretreated for 15 min with 200 nM of the phosphatidyl inositol (PI) 3¢-kinase inhibitor wortmannin prior
to stimulation with insulin or VO(acac)2. Wortmannin
blocked insulin and VO(acac)2 stimulated phosphorylation of GSK-3b (Fig. 8a), as well as the activation of
glycogen synthase (Fig. 8b). In contrast, wortmannin
had no influence on insulin or VO(acac)2 induced
tyrosine phosphorylation (Fig. 8c). Cumulatively, these
results strongly suggest that VO(acac)2 enhanced glycogen synthase activation through the insulin receptor
and initiation of classical, PI 3¢-kinase dependent
insulin signaling cascade rather than through direct
influence on glycogen synthase or the kinase or
phosphatase enzymes that control its phosphorylation
state.
Influence of VO(acac)2 on insulin-resistant 3T3-L1
adipocytes
In type II diabetes, insulin resistance is characterized by
a decrease in both sensitivity and responsiveness of tissues to the circulating hormone [41–43]. The results of
previous investigations have indicated that 18-h treatment of 3T3-L1 adipocytes with low-dose (1 nM) insulin
results in the induction of insulin resistance [30]. We next
examined the influence of VO(acac)2 on glucose metabolism in insulin-resistant 3T3-L1 adipocytes.
In Fig. 9a, it is seen that the extent of insulin-stimulated tyrosine phosphorylation of IRb and IRS1 in
insulin-resistant adipocytes was markedly reduced
compared with that in control cells. Incubation of the
insulin-resistant adipocytes with 0.25 mM VO(acac)2
restored the phosphorylation of insulin receptor and
IRS1 to levels observed in normal cells, even in the
882
resistant cells, VOSO4 had little effect on insulin action
in either type of 3T3-L1 adipocyte (Fig. 10).
Discussion
Chemical and structural stability of VO2+ chelates
in solution
Fig. 7 Effect of VO(acac)2 on insulin-stimulated glycogen synthesis
and glycogen synthase (GS) activity. Replicate 12-wells of 3T3-L1
adipocytes were serum-starved for 3 h. The cells were then stimulated in triplicate with the indicated concentration of insulin, in the
presence of 0.25 mM VO(acac)2 or VOSO4 for 15 min. a Cell lysates
were prepared, and GS activity was measured in vitro, in the
absence and presence of 10 mM glucose-6-phosphate. b For measurement of glycogen synthesis, 2 lCi of 14C-glucose was added to
all wells after the 15-min stimulation with insulin and chelates. After
30 min, the cells were washed and glucose incorporation into
glycogen was determination. VO(acac)2 significantly increased GS
activation by insulin, resulting in synergistic enhancement of
insulin-stimulated glycogen synthesis
absence of added insulin. Similarly, Fig. 9b illustrates
that insulin-mediated phosphorylation of GSK-3b was
significantly reduced in insulin-resistant 3T3-L1 adipocytes compared with that in control cells. However,
VO(acac)2 restored phosphorylation of GSK-3b in
insulin-resistant cells to near maximal levels.
Figure 10a correspondingly compares the influence of
VO(acac)2 and VOSO4 on the incorporation of glucose
into glycogen in control and insulin-resistant 3T3-L1
adipocytes. In insulin-resistant cells, the effect of insulin
on glycogen synthesis was noticeably decreased compared with that in normal cells. Treatment of insulinresistant adipocytes with VO(acac)2 in the presence and
absence of insulin restored glycogen synthesis activity to
nearly control levels. Interestingly, while VO(acac)2
acted synergistically with insulin in control and insulin-
Schieven et al. [44] have shown that 10–25 lM VO(malto)2
acts as an inhibitor of protein tyrosine phosphatases
altering signal transduction during induction of B cell
apoptosis. Peters et al. [22] concluded on the basis of
NMR and X-ray studies that the competitive inhibition
of VO(malto)2 against protein tyrosine phosphatases can
be attributed to the unliganded VO2+ cation extracted
from the intact complex. On this basis, the influence
of VO(malto)2 on glucose metabolism appears not to
derive from the intact metal-chelated complex but rather
from VO2+ binding to macromolecular components of
insulin signal transduction pathways more tightly than
to the maltolato ligands. Our observations of low insulin-enhancing activity by VO(malto)2 in Figs. 2 and 3
comparable to that observed for inorganic VO2+ are in
accord with these conclusions, suggesting that albumin
and transferrin similarly extract VO2+ from a significant
fraction of the intact, chelated complex. In contrast, the
intensity of tyrosine phosphorylated proteins elicited by
VO(OPT)2 in Figs. 2 and 3 was more marked than for
VO(malto)2. By EPR studies the structural intactness of
VO(OPT)2 appears not to be disrupted in the presence of
albumin as severely as is that of VO(malto)2 (D. Mustafi,
M.W. Makinen, unpublished observations).
We conclude, therefore, that the relative ordering of
intensities of tyrosine phosphorylated proteins in Fig. 2
parallels the chemical stability of the VO2+ chelates, as
evidenced by the results in Fig. 5, and their ability
to remain intact in the midst of proteins. Therefore,
the insulin-enhancing activity of VO(malto)2 in these
metabolic assays must be ascribed to a mixture of
effects resulting from albumin:VO2+complexes, albumin: VO(malto)2 complexes, and possibly hemichelate
forms of the complex bound to serum albumin. The
insulin-enhancing activity of VO(OPT)2 is due to an
albumin-bound form of the VO2+ chelate and the free
complex as a minimum number of species. On the
other hand, VO(acac)2 binds to serum albumin as an
intact chelate in 1:1 stoichiometry [10] and elicits the
most intense pattern of tyrosine phosphorylated proteins. Because the insulin-mimetic action of VO(acac)2
in these assays is best ascribed to the albumin: VO
(acac)2 adduct as the predominant species, we employed only VO(acac)2 for further mechanistic studies
because of its inherently greater stability and because it
is associated with the highest activity measured
according to the intensity of phospho-tyrosine immunoblots.
883
Fig. 8 Wortmannin (wort)
inhibition of the influence of
VO(acac)2 on GSK-3b and GS.
Replicate 12-well dishes of
3T3-L1 adipocytes were serumstarved for 2.5 h. The cells were
pretreated for 15 min in the
absence and presence of
200 nM wort, prior to the
addition of 10 nM
insulin ± 0.25 mM VO(acac)2
or VOSO4, as indicated. a After
5 min, lysates prepared and
analyzed by anti-pGSK-3b
immunoblotting. b After
15 min of stimulation, GS
activity was measured in vitro,
in the absence and presence of
10 mM glucose-6-phosphate. c
After 5 min, lysates were
prepared and analyzed by antiphosphotyrosine
immunoblotting. GS activity
results are representative of
three independent experiments,
each performed in duplicate,
while immunoblots are
representative of three to four
independent experiments
Molecular locus of the insulin-enhancing action
of VO(acac)2
Suppression of insulin-induced signaling by the classical inhibitor wortmannin occurs at the level of PI 3¢kinase, the first component of the enzyme cascade to
transduce the effects of IRS1 when activated through
the kinase activity of the insulin receptor [37]. Thus,
although increased phosphorylation of PKB and
GSK-3b and increased glycogen synthase activity were
observed upon addition of VO(acac)2 to 3T3-L1
adipocytes, as observed through Figs. 6 and 7, inhibition of these effects by wortmannin, as demonstrated
in Fig. 8, rules out significant direct action of the
VO2+ chelate on these components of the insulinsignaling pathway. In view of the increased tyrosine
phosphorylation of immunoprecipitated IRb and IRS1
in Fig. 2, we conclude that the insulin-enhancing action of VO(acac)2 is associated with insulin receptor
activation. However, it not certain whether VO(acac)2
directly stimulates insulin receptor tyrosine kinase
activity, or whether it acts indirectly through activation of other tyrosine kinases or inhibition of tyrosine
phosphatases. Determination of the molecular pathways through which VO(acac)2 exerts its insulin-like
effects will require further study.
The group of Shechter [8, 9] ascribe the greater potency
of VO(acac)2 over that of VOSO4 in stimulating
884
Fig. 9 Restoration of IRS1,
IRb, and GSK-3b
phosphorylation by VO(acac)2
in insulin-resistant 3T3-L1
adipocytes. 3T3-L1 adipocytes
were preincubated in the
absence (basal) or presence of
1 nM insulin for 15 h. The next
day, the cells were serumstarved for 3 h, prior to a 5-min
treatment with 0.25 mM
VO(acac)2 in the absence or
presence of 1 or 10 nM insulin.
Lysates were prepared, and
analyzed by antiphosphotyrosine (a) or antipGSK-3b (b) immunoblotting.
Bands corresponding to IRS1,
IRb and pGSK-3b are indicated
by arrows. All results are
representative of two to four
independent experiments
lipogenesis purely to an insulin-independent pathway and
increased stability of the VO2+ chelate against oxidation.
Since they employed only primary rat adipocytes in their
experiments [8, 9, 27, 45, 46], one potential source of
this apparent contradiction is that primary adipocytes
differ metabolically from cultured 3T3-L1 adipocytes
with respect to the effects of vanadium compounds: It is
well known that the main metabolic action of primary
adipocytes is to convert glucose into lipids and fatty acids,
while the primary metabolic flux in 3T3-L1 adipocytes is
the conversion of glucose into glycogen [47]. It is, in
our estimation, unlikely that these contradictory interpretations of experimental results have their origin in
differences in metabolic fluxes between the two cell lines.
The group of Shechter [9] demonstrated that inorganic
VO2+ is noncompetitively inhibitory against purified
insulin receptor protein tyrosine kinase and that it is
inhibitory to insulin receptor autophosphorylation. In
these studies, the group of Shechter investigated the
effects only of inorganic VO2+ added as VOSO4 and
inorganic vanadate on the insulin receptor. At no point in
their experiments did they directly test the influence of
VO(acac)2 on insulin receptor tyrosine kinase activity.
As demonstrated in Figs. 2 and 3, VOSO4 added to
3T3-L1 adipocytes did not elicit tyrosine phosphorylation of IRb or of IRS1 above basal conditions in the
presence or absence of insulin, in contrast to the clear
dose-dependent and insulin-synergistic influence of
VO(acac)2 shown in Fig. 4. Moreover, through Fig. 8 we
have demonstrated not only that VO(acac)2-enhanced
glycogen synthase action and VO(acac)2-enhanced
phosphorylation of GSK-3b were wortmannin-sensitive,
but also that wortmannin in the same adipocyte cells did
not inhibit insulin-activated or VO(acac)2-facilitated
tyrosine phosphorylation of IRb or of IRS1. Furthermore, the reduced levels of tyrosine phosphorylation of
IRb and IRS1 elicited by VO(malto)2 and VO(OPT)2, as
illustrated in Figs. 2 and 3, are fully consistent with their
decreased chemical stability as intact VO2+ chelates, as
shown through Fig. 5. These results collectively argue
strongly that VO(acac)2 promotes the tyrosine phosphorylation of the insulin receptor resulting in initiation of
several insulin-senstive signaling pathways. The emphasis
by the group of Shechter [9, 27, 33, 45, 46] that the insulinlike effects of vanadium compounds occur entirely
through a cytosolic, insulin-receptor-independent protein tyrosine kinase enzyme was made without testing
directly VO2+ chelates in which the organic ligand
exhibits high affinity for the VO2+ ion, such as VO
(acac)2.
Peroxovanadates (of oxidation state V) are associated
with greatly increased phosphorylation of the insulin
receptor owing to their capacity for irreversible oxidation of the active-site cysteine residue of protein phosphatases [48, 49]. There is, however, no resemblance of
the insulin-like action of VO(acac)2 facilitating tyrosine
phosphorylation of IRb and IRS1 described through
these investigations with the action of peroxovanadates.
Vanadates (of oxidation state V) are known to exhibit a
strong tendency to bind to sulfhydryl groups, while
VO2+ (of oxidation state IV) shows low affinity for
sulfur-donor ligands. For instance, the low affinity of
885
Need for development of specific insulin-mimetic
compounds
Fig. 10 Effect of VO(acac)2 on glycogen synthesis and GS activation in insulin-resistant 3T3-L1 adipocytes. 3T3-L1 adipocytes were
made insulin-resistant as in Fig. 9, serum-starved for 3 h, and then
stimulated in the absence or presence of 10 nM insulin. Results are
compared for 0.25 mM VO(acac)2 (A) and VOSO4 (S). a After
15 min, 2 lCi of 14C-glucose was added to all wells. The cells were
placed on ice 30 min later, washed three times with cold phosphatebuffered isotonic saline and glucose incorporation into glycogen was
determined. b The cells were stimulated for 15 min with the agents,
and then cellular lysates were prepared. GS activity was measured in
vitro. VO(acac)2 significantly stimulated glycogen synthesis and
increased GS activity in insulin-resistant cells, in both the absence
and the presence of insulin. The results are the average of three
independent experiments, performed in triplicate or duplicate
VO2+ for sulfhydryl groups is readily demonstrated
through the study by Cornman et al. [50] showing that
VO2+ added to a synthetic polypeptide analog of the
active site of protein phosphatase 1B was bound to a
histidine and a serine residue rather than to the cysteine
residue. Although VO2+ extracted from VO(malto)2 has
been implicated as a reversible inhibitor of protein
phosphatases [22], the stability of VO(acac)2, as shown
in these studies, is significantly greater. It is, therefore,
unclear if potentiation of tyrosine phosphorylation of
the insulin receptor facilitated by VO(acac)2 occurs only
through inhibition of phosphatase action, and further
studies will be needed to examine the relative contributions of kinase activation and phosphatase inhibition to
the enhancement of tyrosine phosphorylation of the
insulin receptor.
Type II diabetes mellitus is a complex metabolic disease
involving defects in both insulin secretion and insulin
action and is influenced by a broad range of genetic and
environmental factors. Although there is still considerable lack of consensus about the primary defect, the
pathogenesis of type II non-insulin-dependent diabetes
mellitus involves progressive development of insulin
resistance and a defect in insulin secretion. Overt disease
marked by hyperglycemia begins when insulin output
from the pancreas fails to meet the requirement for
insulin as a result of insulin resistance [41, 42]. The
requirement for insulin at the cell surface begins with
binding of insulin to the insulin receptor, initiating
autophosphorylation of the b-subunits through activation of its intrinsic tyrosine kinases. Subsequent transphosphorylation of IRS proteins and activation of
downstream signaling molecules continue the spectrum
of biological responses associated with the physiology
of this important polypeptide hormone. While most
patients with type II diabetes initially respond to insulin
secretogues, agents that facilitate pancreatic output of
the hormone, a significant fraction show defects in
insulin signaling. Thus, development of orally active
agents that enhance or mimic the action of insulin could
lead to additional routes for therapy; also, such agents
could be advantageous in the treatment of patients with
type I diabetes who depend on exogenous insulin for
metabolic control.
It is evident that the functioning of the receptor
tyrosine kinase activity is essential for the biological
effects of insulin, and development of orally active, small
molecules that could facilitate the action of insulin at the
level of the insulin receptor enhancing its autophosphorylative and tyrosine kinase activities could be of
pharmaco-therapeutic value. In our studies we have
demonstrated that the action of VO(acac)2 was synergistic with insulin and that the synergism was observable
generally only under low concentrations of insulin. This
result helps to explain the observations of others [5, 6]
that VO2+ compounds are associated with insulinenhancing activity only in diabetic laboratory animals
(in which there is a deficit of circulating insulin hormone
or systemic insulin resistance). Our observations that
high concentrations of insulin mask the synergism
indicate that the insulin-like action of VO2+ compounds
is not restricted to only diabetic tissue. In this regard
VO2+ chelates with insulin-enhancing activity can be
viewed as potential reagents for reducing the level of
insulin to maintain euglycemia. Development of the
therapeutic potential of VO2+ chelates, such as VO
(acac)2, therefore, may be advantageous as insulinenhancing agents also in type I diabetes. To this end,
identification of structural and kinetic factors that
ensure specificity in insulin-enhancing effects will be
important. It is, therefore, of interest to note that the
origin of virtually all of the tyrosine phosphorylated
886
proteins that are observed through immunoblotting
experiments, such as in Figs. 2 and 3, including control,
basal conditions [51, 52], have not been identified.
Assignment of their cellular origins and the dependence
of their expression on insulin binding to its receptor in
the membrane may become a sensitive probe to guide
design of specificity of therapeutic reagents.
Acknowledgements This work was supported by grants of the
National Institutes of Health (DK57599 and DK20959). M.J.B.
is a recipient of a Career Development Award of the American
Diabetes Association.
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