Joseph Murdaca

Methylglyoxal Impairs the Insulin Signaling Pathways Independently of the Formation of Intracellular Reactive Oxygen Species

Nonenzymatic glycation is increased in diabetes and leads to elevated levels of advanced glycation end products (AGEs), which link hyperglycemia to the induction of insulin resistance. In hyperglycemic conditions, intracellularly formed α-ketoaldehydes, such as methylglyoxal, are an essential source of intracellular AGEs, and the abnormal accumulation of methylglyoxal is related to the development of diabetes complications in various tissues and organs. We have previously shown in skeletal muscle that AGEs induce insulin resistance at the level of metabolic responses. Therefore, it was important to extend our work to intermediates of the biosynthetic pathway leading to AGEs. Hence, we asked the question whether the reactive α-ketoaldehyde methylglyoxal has deleterious effects on insulin action similar to AGEs. We analyzed the impact of methylglyoxal on insulin-induced signaling in L6 muscle cells. We demonstrate that a short exposure to methylglyoxal induces an inhibition of insulin-stimulated phosphorylation of protein kinase B and extracellular-regulated kinase 1/2, without affecting insulin receptor tyrosine phosphorylation. Importantly, these deleterious effects of methylglyoxal are independent of reactive oxygen species produced by methylglyoxal but appear to be the direct consequence of an impairment of insulin-induced insulin receptor substrate-1 tyrosine phosphorylation subsequent to the binding of methylglyoxal to these proteins. Our data suggest that an increase in intracellular methylglyoxal content hampers a key molecule, thereby leading to inhibition of insulin-induced signaling. By such a mechanism, methylglyoxal may not only induce the debilitating complications of diabetes but may also contribute to the pathophysiology of diabetes in general.

Evidence for a Differential Interaction of SHC and the Insulin Receptor Substrate-1 (IRS-1) with the Insulin-like Growth Factor-I (IGF-I) Receptor in the Yeast Two-hybrid System

Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr-1131, −1135, and −1136) of the IGF-IR, and (iv) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR.

Grb10 Prevents Nedd4-mediated Vascular Endothelial Growth Factor Receptor-2 Degradation

One of the cellular mechanisms used to prevent continuous and enhanced activation in response to growth factors is the internalization and degradation of their receptors. Little is known about the molecular mechanisms involved in vascular endothelial growth factor receptor-2 (VEGF-R2) degradation. In a previous work, we have shown that the adaptor protein Grb10 is a positive regulator of the VEGF signaling pathway. Indeed, VEGF stimulates Grb10 expression, and Grb10 overexpression induces an increase in the amount and the tyrosine phosphorylation of VEGF-R2. In the present manuscript, we demonstrate that Grb10 stimulates VEGF-R2 expression by inhibiting the Nedd4-mediated VEGF-R2 degradation. First, we show that proteasome inhibition by MG132 induces an increase in VEGF-R2 amount, and that VEGF-R2 is ubiquitinated in response to VEGF. Expression of Nedd4, a HECT domain-containing ubiquitin ligase, induces the disappearance of VEGF-R2 in cells, suggesting that Nedd4 is involved in VEGF-R2 degradation. To determine whether Nedd4 directly ubiquitinates VEGF-R2, we expressed a ubiquitin ligase-deficient mutant Nedd4C854S. In the presence of Nedd4C854S, VEGF-R2 is expressed and ubiquitinated. These results suggest that VEGF-R2 is ubiquitinated but that Nedd4 is not involved in this process. Finally, we show that Grb10 constitutively associates with Nedd4. Co-expression of Nedd4 and Grb10 restores the expression of VEGF-R2, suggesting that Grb10 inhibits the Nedd4-mediated degradation of VEGF-R2. In this study, we show that Grb10 acts as a positive regulator in VEGF-R2 signaling and protects VEGF-R2 from degradation by interacting with Nedd4, a component of the endocytic machinery.

PPARβ Activation Induces Rapid Changes of Both AMPK Subunit Expression and AMPK Activation in Mouse Skeletal Muscle

AMP-activated protein kinases (AMPK) are heterotrimeric, αβγ, serine/threonine kinases. The γ3-AMPK subunit is particularly interesting in muscle physiology because 1) it is specifically expressed in skeletal muscle, 2) α2β2γ3 is the AMPK heterotrimer activated during exercise in humans, and 3) it is down-regulated in humans after a training period. However, mechanisms underlying this decrease of γ3-AMPK expression remained unknown. We investigated whether the expression of AMPK subunits and particularly that of γ3-AMPK are regulated by the PPARβ pathway. We report that PPARβ activation with GW0742 induces a rapid (2 h) and sustained down-regulation of γ3-AMPK expression both in mouse skeletal muscles and in culture myotubes. Concomitantly, phosphorylation levels of both AMPK and acetyl-coenzyme A carboxylase are rapidly modified. The γ3-AMPK down-regulation is also observed in muscles from young and adult transgenic mice with muscle-specific overexpression of peroxisome proliferator-activated receptor β (PPARβ). We showed that γ3-AMPK down-regulation is a rapid physiological muscle response observed in mouse after running exercise or fasting, two situations leading to PPARβ activation. Finally, using C2C12, we demonstrated that dose and time-dependent down-regulation of γ3-AMPK expression upon GW0742 treatment, is due to decrease γ3-AMPK promoter activity.

A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development

Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor β (PPARβ) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARβ activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4−CD8− double-negative stage 4 (DN4) thymocytes. These results support a model where PPARβ activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αβ T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells.

α-Lipoic acid up-regulates expression of peroxisome proliferator-activated receptor β in skeletal muscle: involvement of the JNK signaling pathway

We hypothesized that α‐lipoic acid (α‐LA) might interact with the transcriptional control of peroxisome proliferator‐activated receptor (PPAR)b in skeletal muscle. Molecular mechanisms were investigated using differentiated C2C12 myotubes treated with α‐LA and/or PPARβ agonist GW0742. In vivo studies with 3‐mo‐old C57Bl6 mice were realized: voluntary wheel running (VWR) training (7 wk), and a 6 wk diet containing (or not) α‐LA (0.25% wt/wt). This last condition was combined with (or not) 1 bout of treadmill exercise (18 m/min for 1 h). Using a reporter assay, we demonstrate that α‐LA is not an agonist of PPARβ but regulates PPARβ target gene expression through an active PPARβ pathway. GW0742‐induced pyruvate dehydrogenase kinase 4 mRNA is potentiated by α‐LA. In C2C12, α‐LA lowers the activation of the JNK signaling pathway and increases PPARβ mRNA and protein levels (2‐fold) to the same extent as with the JNK inhibitor SP600125. Similarly to VWR training effect, PPARβ expression increases (2‐fold) in vastus lateralis of animals fed an α‐LA‐enriched diet. However, α‐LA treatment does not further stimulate the adaptive up‐regulation of PPARβ observed in response to 1 bout of exercise. We have identified a novel mechanism of regulation of PPARβ expression/action in skeletal muscle with potential physiologic application through the action of α‐LA, involving the JNK pathway.—Rousseau, A.‐S., Sibille, B., Murdaca, J., Mothe‐Satney, I., Grimaldi, P. A., Neels, J. G., α‐Lipoic acid up‐regulates expression of peroxisome proliferator‐activated receptor b in skeletal muscle: involvement of the JNK signaling pathway. FASEB J. 30, 1287–1299 (2016). www.fasebj.org

Peroxisome Proliferator Activated Receptor Beta (PPARβ) activity increases the immune response and shortens the early phases of skeletal muscle regeneration

Peroxisome Proliferator-Activated Receptor Beta (PPARβ) is a transcription factor playing an important role in both muscle myogenesis and remodeling, and in inflammation. However, its role in the coordination of the transient muscle inflammation and reparation process following muscle injury has not yet been fully determined. We postulated that activation of the PPARβ pathway alters the early phase of the muscle regeneration process, i.e. when immune cells infiltrate in injured muscle. Tibialis anteriors of C57BL6/J mice treated or not with the PPARβ agonist GW0742 were injected with cardiotoxin (or with physiological serum for the contralateral muscle). Muscle regeneration was monitored on days 4, 7, and 14 post-injury. We found that treatment of mice with GW0742 increased, at day 4 post-damage, the recruitment of immune cells (M1 and M2 macrophages) and upregulated the expression of the anti-inflammatory cytokine IL-10 and TGF-β mRNA. Those effects were accompanied by a significant increase at day 4 of myogenic regulatory factors (Pax7, MyoD, Myf5, Myogenin) mRNA in GW0742-treated mice. However, we showed an earlier return (7 days vs 14 days) of Myf5 and Myogenin to basal levels in GW0742- compared to DMSO-treated mice. Differential effects of GW0742 observed during the regeneration were associated with variations of PPARβ pathway activity. Collectively, our findings indicate that PPARβ pathway activity shortens the early phases of skeletal muscle regeneration by increasing the immune response.

P21 L’environnement « redox » détermine t-il les effets géniques de PPARbeta dans le muscle ?

Introduction Une activite physique aerobie, moderee et reguliere, permet de reduire l’incidence du diabete de type II chez des sujets « metaboliquement » exposes, en augmentant le catabolisme lipidique et les capacites antioxydantes musculaire. Ce remodelage oxydatif peut etre induit chez la souris par l’activation de la voie PPARbeta. Nous avons montre que l’expression de PPARbeta etait diminuee dans deux situations physiologiques d’exposition au stress oxydant (l’exercice intense « aigu » et le vieillissement). Objectif Montrer que le statut redox des cellules musculaires determine les effets geniques de PPARbeta (potentiel contractile et metabolique) par une modulation de l’activation des voies de signalisation « redox-sensibles ». Materiels et methodes Nous avons maintenu des myotubes C2C12 et C2C12 surexprimant PPARbeta (C2C12 – PPARbeta) dans des conditions normales ou stressantes (TNF–, LPS). Nous avons co-traite les cellules par un antioxydant puissant : l’acide –-lipoique (ALA) et par du GW0742 (agoniste specifique de PPARbeta). Par l’ajout d’inhibiteurs et d’activateurs, nous avons determine les voies de signalisation affectees par ces conditions. L’expression genique et proteique de PPARbeta et de ses genes cibles a ete mesuree. Resultats Nos resultats montrent que l’activite de NFKB et les concentrations de glutathion total (GSHtot) sont PPARbeta dependantes. L’ALA augmente l’expression genique et proteique de PPARbeta d’un facteur 2 dans les C2C12 et permet l’acquisition d’un statut en GSHtot similaire a celui des C2C12-PPARbeta. Le traitement a l’ALA des C2C12 a des effets geniques communs et additifs a ceux d’un traitement au GW0742 sans qu’il ne soit demontre que l’ALA soit un activateur de PPARbeta. Ces effets semblent passer par une modulation de l’activite de kinases du stress. Conclusion De mimer les effets « benefiques » d’une activation de PPARbeta permet d’envisager des nouvelles strategies therapeutiques et preventives du diabete de type II en maitrisant les conditions de stress cellulaire.