Mechanism of Insulin Mimetic Action of Peroxovanadium Compounds

Chapter 25

Mechanism of Insulin Mimetic Action of Peroxovanadium Compounds Downloaded by MICHIGAN STATE UNIV on February 20, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch025

Β. I. Posner, C. R. Yang, and A. Shaver Departments of Medicine and Chemistry, McGill University, Montreal, Quebec H3A 2B2, Canada The peroxovanadium compounds (pVs) have been shown to be insulin mimetic. They achieve this by potently activating the insulin receptor kinase (IRK) in the complete absence of insulin. This mechanism of insulin mimesis differs from that of vanadate (V) whose site(s) of action appear(s) to be distal to the IRK. Evidence to date indicates that pVs activate the IRK by inhibiting an intimately associated phosphotyrosine phosphatase (PTP) which normally prevents IRK autophosphorylation. When this enzyme is inhibited the IRK is able to autoactivate in the absence of any restraint to autophosphorylation. Both V and pVs are potent inhibitors of PTPs. However V, unlike pVs, is readily chelated and hence in biological systems is less available for effecting PTP inhibition. The inhibition of PTP by pVs appears to involve the irreversible oxidation of a key cysteine residue in the catalytic site of the enzyme. In biological systems pV appears to inhibit, relatively selectively, the PTP associated with IRKs which are intracellular (ie. within the endosomal system of the cell). This observation has supported the view that important aspects of IRK transmembrane signaling occur within the cell. The efficacy of pVs to lower blood glucose levels in both normal and diabetic animals indicates that a key molecular target for the development of insulin mimetic drugs is the IRK-associated PTP.

Insulin action begins with the binding of insulin to its receptor; a heterotetrameric protein made up of two α subunits, and two transmembrane β subunits whose cytosolic domains contain tyrosine kinase activity. Following insulin binding to the α subunit of the receptor the β subunit undergoes autophosphorylation on tyrosine residues leading to activation of the insulin receptor kinase (IRK) towards exogenous substrates. Numerous studies have shown that this activation process is the key to insulin signaling (/). Indeed mutated IRK molecules, unable to undergo

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©1998 American Chemical Society In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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autophosphorylation and autoactivation, cannot entrain the insulin signaling sequence (2). Following the binding of insulin to the ERK there is rapid internalization of the insulin-IRK complex into a tubulovesicular organelle within the cell, known as the endosomal system or endosomes (ENs) (3). Substantial evidence now exists establishing this step as part of the mechanism involved in the insulin signaling sequence (4). The Discovery Of pV Complexes. Studies in the early to mid 1980s established vanadate as an insulin mimetic agent in vitro and in vivo. We subsequently showed that the combination of vanadate and H 0 activated the IRK and promoted insulin signaling in a synergistic manner (5), and that this was due to peroxovanadium (pV) complexes which formed on combining vanadate and H2O2 (6). Different pVs, synthesized by incorporating different ancillary ligands into the complex, could be readily differentiated from one another using V N M R . The pV complexes were crystallizable from solution, stable at neutral pH in the absence of light and far more potent as insulin mimetics than vanadate (7). Subsequent to our discovery of the insulin like activity of pVs these compounds were shown to mimic a wide range of insulin effects (8-9).

Downloaded by MICHIGAN STATE UNIV on February 20, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch025

2

2

5 1

pVs Inhibit IRK-Associated PTP(s). In subsequent studies we evaluated the mechanism by which pVs activate the IRK in the complete absence of insulin (7). Initial studies showed that incubating partially purified IRKs with pVs did not activate the IRK in contrast to what is seen on incubation with insulin (6). When ENs, isolated from rats previously treated with insulin, were incubated with A T P we could demonstrate increasing tyrosine phosphorylation of the IRK which spontaneously diminished in parallel with the depletion of ATP in the incubation mixture. Coincubation with pV augmented the level of IRK tyrosine phosphorylation and completely abrogated the diminution of phosphorylation as ATP levels diminished during the incubation (10). Thus pVs inhibit a dephosphorylation process affecting the IRK, and we inferred that this was due to the inhibition of an IRK-associated phosphotyrosine phosphatase (PTP) (10). It has subsequently been shown that pVs constitute the most potent class of PTP inhibitors described to date (7). The Mechanism of PTP Inhibition Effected by pVs. To study the mechanism by which pVs inhibit PTPs we evaluated their action on PTP1C (SHPTP1). To do this we prepared highly purified enzyme for our analyses. The coding region of this enzyme was cloned into the plasmid pET-3C which was used to transform E.Coli BL-21 (DE3) which carries the T7 polymerase gene under the control of the lacUV5 promoter. The expression of PTP1C was induced by adding 20 u M isopropylthiogalactoside (IPTG) to the bacterial culture medium, and the cells were subsequently harvested by centrifugation and broken by sonication in the presence of high salt buffer containing mild detergent. Enzyme activity at each step in the purification was evaluated. To assure stability the enzyme was kept in the presence of 1 m M EDTA and 2mM dithiothreitol

In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

318 (DTT). PTP1C was purified to homogeneity in a 3-step procedure, involving precipitation by ammonium sulphate (30-50%) followed by chromatography on DEAE-Sephadex A-50 (elution with 0-0.4 NaCl gradient at pH 7.5) and then SPSephadex C-50 (elution with 0-0.5 M NaCl gradient at pH7.0). This last step yielded a peak of activity, which migrated as a single protein band on gel electrophoresis indicating that attainment of a high level of purity. This was confirmed by establishing that the amino acid composition of the protein band conformed precisely to the predicted composition based on previous studies (77). Figure 1 illustrates the inhibitory effect of bpV (pic), bpV (phen), and vanadate on PTP1C activity. As can be seen 50% inhibition (I ) of PTP1C activity was produced at a concentration of 2.5 χ 10" M bpV (pic), 2 χ 10' M bpV (phen), and 6 χ ΙΟ" M vanadate in the absence of EDTA. Whereas 1 m M EDTA had no effect on the inhibitory potency of the pV compounds it reduced the I of vanadate to 3 χ 10' M . We have routinely observed that vanadate has an inhibitory potency 0.001% that of pVs on IRK-associated PTP activity in ENs. Thus in complex mixtures the chelation of vanadate is probably responsible for a substantial part of its reduced potency compared to pVs as an inhibitor of PTPs. Further studies of 18 different pVs showed an I ranging from 2 χ 10" to 2 χ ΙΟ" M . The I o of the tungstate complex bpW(pic) was 3 χ 10" M and that of the molybdate complex bpMo (pic) was 7 χ 10" M . Table I illustrates that the inhibitory effect of vanadate but not that of pV can be reversed by a strong chelating agent. Thus premixing desferoxamine Β with either vanadate or pV prevented only the inhibitory activity of the former and not the latter. This corifirms the above data indicating that vanadate is readily chelated and thereby inactivated but pV is not. When vanadate or pV were first added to the enzyme so as to effect complete inhibition and desferoxamine was subsequently added only inhibition by vanadate was reversed indicating that it forms a reversible association with the enzyme. The failure to reverse the inhibitory effect of pV may reflect irreversibility of inhibition but can be explained by the failure of desferoxamine to chelate this compound. To evaluate the possibility that pV promotes irreversible inhibition of PTP1C we incubated the enzyme with vanadate or bpV (phen) and at various times thereafter added DTT and E D T A to block their interaction with PTP1C (Fig. 2). DTT acts to convert pV compounds to vanadate; whereas EDTA, as shown above and elsewhere (72) chelates free vanadate and renders it unavailable to interact with the enzyme. As can be seen the inhibition effected by vanadate is virtually completely reversible. In contrast that effected by bpV (phen) became irreversible in a time and temperature dependent manner. The rate of inactivation evoked by bpV (phen) was considerably accelerated by incubating the mixture at 22° C at which temperature it was essentially complete by 10 mins of incubation. Previous work has shown that PTP activity involves a critical cysteine residue at the active site of the molecule. It has been shown that pV compounds oxidize this residue in the enzyme PTP1B (13). This correlated with the inactivation of PTP1B and hence represents the likely reason for that inactivation. So too in our system we observed that cysteine residues were oxidized by exposure to bpV (phen) under conditions which led to the irreversible inactivation of the enzyme (Table Π). We 50

8

7


7

50

3

7

8

50

5

8

7



319

Log inhibitor concentration (M) Figure 1: Inhibition of PTP-1C By Vanadate (V) and Peroxovanadium Compounds. PTP-1C ( 7 ng ), purified as briefly described in the text, was incubated with vanadate (V) in the presence ( • ) or absence (V) of 1 m M EDTA, bpV(pic) (0) or bpV(phen) ( · ) in 25 m M phosphate buffer, pH 7.4 containing 0.01% of Bovine Serum Albumin at 4 ° £ for 10 min. PTP enzyme activity was subsequently measured by adding P-labelled poly (GluTyr) (10 μΜ) to the mixture and evaluting its dephosphorylation. P - poly (Glu-Tyr) was prepared by incubating unlabeled material with the activated IRK and P - A T P for 1 hr. at 37°C. The percentage inhibition was calculated based on PTP-1C activity in the absence of inhibitors. 3

32

32



320

Table I. Reversal by Desferoxamine Β of the Inhibition of PTP-1C by Vanadate but not pV.

Inhibitor

Plus Desferoxamine Β Pre Post

None

-

100

+ +

1.5 98 90

+

0.5 1.5 1.5

Na V0 3

4

bpV(phen)

-

+

Enzvme Activity (% Control)

PTP-1C was incubated with either 1 m M Vanadate or 10 μΜ bpV(phen) in 25 m M phosphate buffer (pH 7.4) - 0.01% Bovine Serum Albumin for 20 mins at 4°C. Desferoxamine (final concentration 10 mM) was added to vanadate or bpV(phen) in some experiments (Pre) or incubated with the inhibited enzyme for 20 mins at 4°C (Post) prior to proceeding with enzyme assays as described in the legend of Figure 1.


321


100

ο

c ο ο

CL C CO

50 0 -J

bpV(phen)

-

+ + - -

Insulin

+

- + -

IR

+ +

+ -

+

M1030

Figure 5: Phosphotyrosine Content of the β-Subunit of the Insulin Receptor (IR) after bpV(phen) and/or Insulin Treatment of Hepatoma Cells Bearing Normal or Mutant (M1030) Kinase Negative IRs. Cells were incubated with 100 n M insulin for 5 min, 0.1 m M bpV(phen) for 20 min, or 0.1 m M bpV(phen) for 15 min before the addition of 100 n M insulin for 5 min. The cells were then solubilized and IRs were partially purified, immunoprecipitated with anti-IR antibodies, subjected to SDSpolyacrylamide gel electrophoresis and immunoblotted with antiphosphotyrosine and anti-IR antibodies as described in detail by Band et al (Molec.Endocrinol., in press) from which this figure was adapted. As can be seen the kinase negative IR ( M l 030) cannot promote β-subunit tyrosine phosphorylation as can the normal IR in the presence of bpV(phen) ± insulin.


326


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30 Η

25Η 20

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Ο υ Ο (Ό ε

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α. TIME (days)

Figure 6 : Effect of Subcutaneous Injections of bpV(phen) and vanadate on Fasting Plasma Glucose Levels in Insulin-Deprived Diabetic Rats. Diabetic rats were deprived of insulin for 16 hours so that their blood glucose levels rose to 20 to 25 m M (normal levels = 5-7 mM). They were then injected subcutaneously with bpV(phen), 36 μιηοΐ/kg body wt. b.i.d. ( • , η = 6); vanadate, 36 μιηοΐ/kg body wt. b.i.d. (0, η = 6); or phosphate-buffered saline, b.i.d. (• , η = 6) for 3 days. A l l injections were withheld from days 4 to 6. Blood glucose determinations and other experimental details are described in detail in Figure 4 of ref.16 from which this figure is adapted. Whereas bpV(phen) injections lowered blood glucose to near-normal levels, an equimolar dose of vanadate was without effect.


327

(phen) no IRK tyrosine phosphorylation was seen. Thus the source of IRK tyrosine phosphorylation following exposure to pVs is the IRK itself. Presumably the low level of intrinsic kinase activity is sufficient to effect autophosphorylation so that the IRK is "bootstrapped" into a state of augmented activation. The slower time course of activation seen following pV, compared to that seen following insulin (7), is consistent with a slow autocatalytic process in which the receptor's active state is augmented as its level of tyrosine phosphorylation increases.


The IRK-Associated PTP(s) as a Therapeutic Target The above studies have shown that pVs act by inhibiting the function of IRKassociated PTP(s) leading to IRK autophosphorylation and activation with the entrainment of downstream insulin signaling. Thus pVs and other agents able to inhibit the IRK-associated PTP(s) should evoke insulin mimesis and hence be potentially applicable as agents for the treatment of Diabetes Mellitus. In recent studies Jean-Francois Yale et al showed that administering pV compounds to rats lowered blood sugar in both normal and diabetic animals. This was recently well illustrated in a study on diabetic B B Wistar rats whose condition resembles that of Type I Diabetes Mellitus (Insulin Dependent Diabetes Mellitus, IDDM) (16). When insulin is withdrawn from these diabetic animals they become hyperglycemic and decline into ketoacidosis and death. As illustrated in Figure 6 giving bpV (phen) by daily intraperitoneal injection to diabetic B B Wistar rats, withdrawn from insulin 24 hours before, resulted in normalization of blood glucose levels. Thus agents directed at the IRK-associated PTP(s) should eventually be able to replace insulin. It is also likely that some of the devised agents will be orally administrable thus precluding the need for treatment by injection as is required when administering insulin. Summary Studies to date have clearly established that pVs act to mimic insulin by activating the IRK and hence entraining the insulin signaling cascade. This activation follows on the inhibition by pVs of IRK-associated PTP(s) which enables the IRK to effect autophosphorylation and hence autoactivation. This sequence appears to differ from that involved in the insulin mimetic effects of vanadate which seems to act primarily at postreceptor site(s) to yield insulin signaling. Study of the mechanism of pV action has uncovered an important mechanism for regulating IRK function (ie. the IRK-associated PTP(s)). It is anticipated that agents targeted against the IRK-associated PTP(s) will eventually be developed to effect insulin signaling in the complete absence of insulin. Literature Cited 1. 2.

Kahn, C. R. Diabetes 1994, 43, 1066-1084. Chou, C. K.; Dull, T. J.; Russell, D. S.; Gherzi, R.; Lebwohl, D.; Ullrich, Α.; Rosen, Ο. M. J. Biol. Chem. 1987 262, 1842-1847.


328 3. 4. 5. 6. 7.


8. 9. 10. 11. 12. 13. 14. 15. 16.

Khan, M. N.; Baquiran, G.; Brule, C.; Burgess, J.; Foster, Β.; Bergeron, J. J. M.; Posner, B.I. J. Biol. Chem. 1989, 264, 12931-12940. Bevan, A.P.; Drake, P.; Bergeron, J.J.M.; Posner, B.I. Trends Endocrinol.Metab. 1996, 7, 13-21. Kadota, S.; Fantus, I. G.; Deragon, G.; Guyda, H. J.; Posner, Β. I. J. Biol. Chem. 1987 262, 8252-8256. Kadota, S.; Fantus, I. G.; Deragon, G.; Guyda, H. J.; Hersh, B.; Posner Β. I. Biochem. Biophys. Res. Commun. 1987, 147, 259-266. Posner, Β. I.; Faure, R.; Burgess, J. W.; Bevan, A. P.; Lachance, D.; Zhang -Sun, G.; Fantus, I. G.; Ng, J. B.; Hall, D. Α.; Lum, B. S.; Shaver, A. J. Biol. Chem. 1994, 269, 4596-4604. Fantus, I. G.; Kadota, S.; Deragon, G.; Foster, B.; Posner, Β. I. Biochem. 1989, 28, 8864-8871. Bevan, A. P.; Burgess, J. W.; Yale, J.-F.; Drake, P. G.; Lachance, D.; Baquiran, G.; Shaver, Α.; Posner, Β. I. Am. J. Physiol. 1995. 268, E60-E66. Faure, R.; Baquiran, G.; Bergeron, J. J. M.; Posner, Β. I. J. Biol. Chem. 1992, 267, 11215-11221. Shen, S.-H.; Bastien, L.; Posner, Β. I.; Chrétien, P. Nature 1991, 352, 736739. Crans, D. C.; Mahroof-Tahif, M.; Keramidas, A. D. Mol. Cell. Biochem. 1995, 153, 17-24. Huyer, G.; Liu, S.; Kelly, J.; Moffat, J.; Payette, P.; Kennedy, B.; Tsaprailis, G.; Gresser, J. J.; Ramachandran, C. J. Biol. Chem. 1997, 272, 843-851. Drake, P. G.; Bevan,A. P.; Burgess, J. W.; Bergeron, J. J. M.; Posner, Β. I.; Endocrinol. 1996, 137, 4960-4968. Bevan, A. P.; Burgess, J. W.; Drake, P. G.; Shaver, Α.; Bergeron, J. J. M.; Posner, Β. I. J. Biol. Chem. 1995, 270, 10784-10791. Yale, J.-F.; Lachance, D.; Bevan, A. P.; Vigeant, C.; Shaver, Α.; Posner, Β. I. Diabetes 1995, 44, 1274-1279.


Mechanism of Insulin Mimetic Action of Peroxovanadium Compounds

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