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Proteomics

Proteomics and PUGNAcity will overcome questioning of insulin resistance induction by non-selective inhibition of O-GlcNAcase.
Vanessa Dehennaut1 and Tony Lefebvre1
1

Accepted Article

CNRS-UMR 8576, Unit of Structural and Functional Glycobiology, IFR 147, University of Lille 1,

59655 Villeneuve d'Ascq, FRANCE Correspondence: Pr. Tony Lefebvre, email: tony.lefebvre@univ-lille1.fr, phone: + 33 3 20 43 47 58, Fax: + 33 3 20 43 65 55

Received: August 19, 2013; Revised: August 19, 2013; Accepted: August 21, 2013

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi:10.1002/pmic.201300363.

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Abstract
Post-translational modifications (PTM) are the ultimate element that perfects the existence and the activity of proteins. Owing to PTM, no fewer than 500 millions biological activities arise from approximately 20,000 protein-coding genes in human. Hundreds PTM were characterized in living beings among which a large variety of glycosylations. Many compounds have been developed to tentatively block each kind of glycosylation so as to study their biological functions but due to their complexity many off-targets effects were reported. Insulin resistance exemplifies this problem. Several independent groups described that inhibiting the removal of O-GlcNAc moieties using PUGNAc, a non-selective inhibitor of the nuclear and cytoplasmic O-GlcNAcase (OGA), induced insulin resistance both in vivo and ex vivo. The development of potent and highly selective OGA inhibitors called into question that elevated O-GlcNAcylation levels are responsible for insulin resistance, these compounds not recapitulating the insulin-desensitizing effect of PUGNAc. To tackle this intriguing problem, a South Korean group recently combined ATP-affinity chromatography and gel-assisted digestion to identify proteins, differentially expressed upon treatment of 3T3-L1 adipocytes with PUGNAc, involved in protein turnover and insulin signaling.

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Keywords: hexosaminidases inhibitors, PUGNAc, insulin signaling, O-GlcNAcylation, ubiquitination.

Accepted Article
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O-GlcNAcylation is a glycosylation found within the nuclear and the cytosolic compartments of cells. In opposition to N- and O-glycosylations, O-GlcNAcylation is simple at the structural point of view. This glycosylation only consists in the transfer of a single N-acetylglucosamine moiety provided by UDP-GlcNAc, the end-product of HBP (hexosamine biosynthetic pathway), through a beta-linkage onto serine and threonine residues [1]. Like phosphorylation, with which it can compete, OGlcNAcylation is reversible since OGT (O-GlcNAc transferase) manages the transfer of the GlcNAc group and OGA (O-GlcNAcase) removes it. Also, like its unsweetened counterpart, O-GlcNAcylation controls fundamental processes, e.g. cell cycle, cell signaling and intracellular trafficking. Deregulation of O-GlcNAc dynamism may be involved in the development of diabetes [1]. However, the development of insulin resistance, the inability of the organism to respond to insulin and a hallmark of type-2 diabetes, in response to an increased O-GlcNAcylation level is controversial. Although the induction of insulin resistance in adipocytes and in skeletal muscles in response to an elevation of O-GlcNAcylation through inhibition of OGA by PUGNAc (O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino-N-phenylcarbamate) appeared assumed, the development of highlyselective OGA inhibitors challenged these findings [2, 3]. Beyond its inhibitory effect on OGA (GH84 Glycoside Hydrolase 84- in CAZy classification [4]), PUGNAc also interferes with the synthesis of GM2 and with - and -N-acetylglucosaminidases belonging to families GH3 and GH89 [3]. This lack of specificity could explain why PUGNAc utilization results in insulin resistance while NButGT, an OGA specific inhibitor, is ineffective in reducing 2-DOG (2-deoxyglucose) uptake [2] such as 6-Ac-Cas (6Acetamido-6-deoxy-castanospermine) [3], even though this last compound also inhibits HexB (GH20). Moreover, low concentrations of PUGNAc did not impair 2-DOG uptake while O-GlcNAc levels remained higher [3]. In the same way, it was shown that adenoviral OGA overexpression or OGT knockdown do not restore the impaired activation of Akt in 3T3-L1 cells pre-incubated with high concentrations of glucose and low doses of insulin [5]. Thereby, whether PUGNAc promotes insulin resistance by elevating O-GlcNAc levels remains unlikely. To tentatively overcome this controversial question, Lee and collaborators [6] identified PUGNAc targets by using the combination of ATP-affinity chromatography and proteomic approaches. The authors followed this strategy for two reasons mainly. Studying a subproteome offers the opportunity to identify very low abundance proteins and consequently avoids their loss in the background of the entire proteome. Secondly, ATP is the most abundant nucleotide-compound in the cell and ATP-binding proteins are major components of signaling pathways including those associated to insulin. Amongst the hundreds identified proteins, 261 are nucleotide (ATP, GTP and NAD+) binding proteins. The proteomic analysis covers more than three quarters of the ATP-binding

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protein families proving the approach that was chosen was efficient. The authors more particularly focused on proteins involved in the ubiquitination pathway. They argue that a defect in insulin signaling in 3T3-L1 adipocytes may be caused by an increased ubiquitination of its components (Fig. 1). A decade ago, it was proposed that actors of the ubiquitin pathway had to be considered as partners of components of the insulin signaling pathway. As an example, the E3-ubiquitin ligase made of SOCS (suppressor of cytokine signaling), elongin C, cullin2/5 and RING-box protein 1 regulates the turnover of IRS (insulin receptor substrate) 1/2 [7]. The ubiquitin-proteasome system is also involved in the regulation of the turnover of the insulin receptor, of insulin itself and of transcription factors driving the expression of insulin-induced genes [8]. Lee and collaborators [6] focused on four proteins involved in the activation of ubiquitination: the ubiquitin-activating enzyme E1, Hsp70 and the AAA-ATPase super family member p97/Vcp (vasolin-containing protein) that were up-regulated by PUGNAc, and Hsp90 that was down-regulated (Fig. 1). Regarding Hsp70, activation of HBP enhances the nuclear translocation of Sp1 and HSF1, leading to the expression of the molecular chaperone [9]. The identification of Hsp70 in the proteomic approach may highlight why cytosolic ubiquitination (and ER ubiquitination via Vcp expression) is increased following PUGNAc treatment. On the other hand, this does not fully justify why PUGNAc but not NButGT nor 6-Ac-Cas induce insulin resistance; the three drugs being OGA inhibitors, thus they had to increase Sp1 and HSF1 nuclear transport. More generally, treatment of adipocytes with PUGNAc reverberates on a global increase of ubiquitination. Our group made the same observation on HepG2 cells a few years ago [10]. We found that heat shock and PUGNAc induced a concomitant burst in O-GlcNAcylation and ubiquitination. The invalidation of OGT using siRNA drastically reduced the ubiquitination levels and the O-GlcNAcylation status of E1, but at this time no direct link between the glycosylation of E1 and its activity has been drawn [10]. The last protein, Hsp90 is necessary for the stability of Akt, a downstream effector of insulin signaling [11]. Lee and collaborators showed that expression and activation of Akt (and Hsp90 expression) is thwarted by PUGNAc, not by 6-Ac-Cas while in both cases O-GlcNAcylation levels were up-regulated [6]. PUGNAc exerts an effect on Hsp90 expression, independent of O-GlcNAcylation processes, that remains to be deciphered. In this issue, Lee and collaborators provide new perspective on how PUGNAc induces insulin resistance independently of an elevation of O-GlcNAcylation status [6]. However, while the authors' data suggest that PUGNAc exerts its effect through ubiquitination of components of the insulin pathway, it is not yet understood how PUGNAc interfere with factors involved in protein turnover. Moreover, in a recent paper, our group demonstrated that inhibiting OGT expression interfere with the insulin receptor expression in HepG2 cells [12]. Thus, it cannot be completely exclude yet that OGlcNAcylation processes do not interfere with insulin resistance whereas we did not explore OGT IR-

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dependent expression in adipocytes. Glucose homeostasis is complex and what is assumed in one specific cell type or tissue cannot be systematically in another one. There is still a long way to go to understand how some small molecules inhibitors and not others exert damages on insulin signaling. If Michael Scofield, the hero of "Prison break", had taken selective OGA inhibitors and not PUGNAc, he could not have planned for jail-break: the series would have stopped prematurely only for a problem of OGA selectivity!

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References [1] Lefebvre, T., Dehennaut, V., Guinez, C., Olivier, S., et al., Dysregulation of the nutrient/stress sensor O-GlcNAcylation is involved in the etiology of cardiovascular disorders, type-2 diabetes and Alzheimer's disease. Biochim. Biophys. Acta 2010, 1800, 67-79. [2] Macauley, M.S., Bubb, A.K., Martinez-Fleites, C., Davies, G.J., Vocadlo, D.J., Elevation of global OGlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance. J. Biol. Chem. 2008, 283, 34687-34695. [3] Macauley, M. S., He, Y., Gloster, T. M., Stubbs, K. A., et al., Inhibition of O-GlcNAcase using a potent and cell-permeable inhibitor does not induce insulin resistance in 3T3-L1 adipocytes. Chem. Biol. 2010, 17, 937-948. [4] Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., et al., The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 2009, 37, D233–238. [5] Robinson, K. A., Ball, L. E., Buse, M. G. Reduction of O-GlcNAc protein modification does not prevent insulin resistance in 3T3-L1 adipocytes. Am. J. Physiol. Endocrinol. Metab. 2007, 292, E884890. [6] Lee, J. E., Park, J. A., Moon, P. G., Baek, M. C., Identification of differentially expressed proteins by treatment with PUGNAc in 3T3-L1 adipocytes through analysis of ATP-binding proteome. Proteomics 2013 (to be defined). [7] Rui, L., Yuan, M., Frantz, D., Shoelson, S., White, M.F., SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J. Biol. Chem. 2002,277, 42394–42398. [8] Rome, S., Meugnier, E., Vidal, H., The ubiquitin-proteasome pathway is a new partner for the control of insulin signaling. Curr. Opin. Clin? Nutr. Metab. Care. 2004, 7, 249-254. [9] Hamiel, C. R., Pinto, S., Hau, A., Wischmeyer, P. E., Glutamine enhances heat shock protein 70 expression via increased hexosamine biosynthetic pathway activity. Am. J. Physiol. Cell. Physiol. 2009, 297, C1509-1519. [10] Guinez, C., Mir, A. M., Dehennaut, V., Cacan, R., et al. Protein ubiquitination is modulated by OGlcNAc glycosylation. FASEB J. 2008, 22, 2901-2911. [11] Basso, A. D., Solit, D. B., Chiosis, G., Giri, B., et al., Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J. Biol. Chem. 2002, 277, 39858-39866. [12] Perez-Cervera, Y., Dehennaut, V., Aquino Gil, M., Guedri, K., et al., Insulin signaling controls the expression of O-GlcNAc transferase and its interaction with lipid microdomains. FASEB J. 2013, 27, fj. 12-217984.

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Figure 1- Mechanism by which PUGNAc may induce insulin resistance. Upon binding to its receptor, insulin activates the PI3K/Akt pathway (for clarity only the insulin receptor, PI3K, PDK and Akt were represented). According to Lee and collaborators [6], PUGNAc upregulates Hsp70, E1 and Vcp, increasing ubiquitination of insulin pathway components and downregulates Hsp90 that in turns reverberates on a decreased-Akt expression and phosphorylation: synergistically, these events should diminish insulin signaling and lead to insulin resistance.

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