Effects of the kinase inhibitor sorafenib on heart, muscle, liver and plasma metabolism in vivo using non-targeted metabolomics analysis.

BACKGROUND AND PURPOSE: The human kinome consists of roughly 500 kinases, including 150 that have been proposed as therapeutic targets. Protein kinases regulate an array of signalling pathways that control metabolism, cell cycle progression, cell death, differentiation and survival. It is not surprising, then, that new kinase inhibitors developed to treat cancer, including sorafenib, also exhibit cardiotoxicity. We hypothesized that sorafenib cardiotoxicity is related to its deleterious effects on specific cardiac metabolic pathways given the critical roles of protein kinases in cardiac metabolism. EXPERIMENTAL APPROACH: FVB/N mice (10 per group) were challenged with sorafenib or vehicle control daily for 2 weeks. Echocardiographic assessment of the heart identified systolic dysfunction consistent with cardiotoxicity in sorafenib-treated mice compared to vehicle-treated controls. Heart, skeletal muscle, liver and plasma were flash frozen and prepped for non-targeted GC-MS metabolomics analysis. KEY RESULTS: Compared to vehicle-treated controls, sorafenib-treated hearts exhibited significant alterations in 11 metabolites, including markedly altered taurine/hypotaurine metabolism (25-fold enrichment), identified by pathway enrichment analysis. CONCLUSIONS AND IMPLICATIONS: These studies identified alterations in taurine/hypotaurine metabolism in the hearts and skeletal muscles of mice treated with sorafenib. Interventions that rescue or prevent these sorafenib-induced changes, such as taurine supplementation, may be helpful in attenuating sorafenib-induced cardiac injury.

Exercise-Induced Alterations in Skeletal Muscle, Heart, Liver, and Serum Metabolome Identified by Non-Targeted Metabolomics Analysis.

BACKGROUND: The metabolic and physiologic responses to exercise are increasingly interesting, given that regular physical activity enhances antioxidant capacity, improves cardiac function, and protects against type 2 diabetes. The metabolic interactions between tissues and the heart illustrate a critical cross-talk we know little about. METHODS: To better understand the metabolic changes induced by exercise, we investigated skeletal muscle (plantaris, soleus), liver, serum, and heart from exercise trained (or sedentary control) animals in an established rat model of exercise-induced aerobic training via non-targeted GC-MS metabolomics. RESULTS: Exercise-induced alterations in metabolites varied across tissues, with the soleus and serum affected the least. The alterations in the plantaris muscle and liver were most alike, with two metabolites increased in each (citric acid/isocitric acid and linoleic acid). Exercise training additionally altered nine other metabolites in the plantaris (C13 hydrocarbon, inosine/adenosine, fructose-6-phosphate, glucose-6-phosphate, 2-aminoadipic acid, heptadecanoic acid, stearic acid, alpha-tocopherol, and oleic acid). In the serum, we identified significantly decreased alpha-tocopherol levels, paralleling the increases identified in plantaris muscle. Eleven unique metabolites were increased in the heart, which were not affected in the other compartments (malic acid, serine, aspartic acid, myoinositol, glutamine, gluconic acid-6-phosphate, glutamic acid, pyrophosphate, campesterol, phosphoric acid, creatinine). These findings complement prior studies using targeted metabolomics approaches to determine the metabolic changes in exercise-trained human skeletal muscle. Specifically, exercise trained vastus lateralus biopsies had significantly increased linoleic acid, oleic acid, and stearic acid compared to the inactive groups, which were significantly increased in plantaris muscle in the present study. CONCLUSIONS: While increases in alpha-tocopherol have not been identified in muscle after exercise to our knowledge, the benefits of vitamin E (alpha-tocopherol) supplementation in attenuating exercise-induced muscle damage has been studied extensively. Skeletal muscle, liver, and the heart have primarily different metabolic changes, with few similar alterations and rare complementary alterations (alpha-tocopherol), which may illustrate the complexity of understanding exercise at the organismal level.

Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation.

BACKGROUND: The arcuate nucleus of the hypothalamus regulates food intake. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is expressed in neuropeptide Y and proopiomelanocortin (POMC) neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. However, the target protein(s) of Asb-4 in these neurons remains unknown. Insulin receptor substrate 4 (IRS4) is an adaptor molecule involved in the signal transduction by both insulin and leptin. In the present study we examined the colocalization and interaction of Asb-4 with IRS4 and the involvement of Asb-4 in insulin signaling. RESULTS: In situ hybridization showed that the expression pattern of Asb-4 was consistent with that of IRS4 in the rat brain. Double in situ hybridization showed that IRS4 colocalized with Asb-4, and both Asb-4 and IRS4 mRNA were expressed in proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons within the arcuate nucleus of the hypothalamus. In HEK293 cells co-transfected with Myc-tagged Asb-4 and Flag-tagged IRS4, Asb-4 co-immunoprecipitated with IRS4; In these cells endogenous IRS4 also co-immunoprecipitated with transfected Myc-Asb-4; Furthermore, Asb-4 co-immunoprecipitated with IRS4 in rat hypothalamic extracts. In HEK293 cells over expression of Asb-4 decreased IRS4 protein levels and deletion of the SOCS box abolished this effect. Asb-4 increased the ubiquitination of IRS4; Deletion of SOCS box abolished this effect. Expression of Asb-4 decreased both basal and insulin-stimulated phosphorylation of AKT at Thr308. CONCLUSIONS: These data demonstrated that Asb-4 co-localizes and interacts with IRS4 in hypothalamic neurons. The interaction of Asb-4 with IRS4 in cell lines mediates the degradation of IRS4 and decreases insulin signaling.

C-terminus of heat shock protein 70-interacting protein-dependent GTP cyclohydrolase I degradation in lambs with increased pulmonary blood flow.

We showed that nitric oxide (NO) signaling is decreased in the pulmonary vasculature before the development of endothelial dysfunction in a lamb model of congenital heart disease and increased pulmonary blood flow (Shunt). The elucidation of the molecular mechanism by which this occurs was the purpose of this study. Here, we demonstrate that concentrations of the endogenous NO synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), are elevated, whereas the NOS cofactor tetrahydrobiopterin (BH(4)) is decreased in Shunt lambs. Our previous studies demonstrated that ADMA decreases heat shock protein-90 (Hsp90) chaperone activity, whereas other studies suggest that guanosine-5'-triphosphate cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the generation of BH(4), may be a client protein for Hsp90. Thus, we determined whether increases in ADMA could alter GCH1 protein and activity. Our data demonstrate that ADMA decreased GCH1 protein, but not mRNA concentrations, in pulmonary arterial endothelial cells (PAECs) because of the ubiquitination and proteasome-dependent degradation of GCH1. We also found that Hsp90-GCH1 interactions were reduced, whereas the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) increased in ADMA-exposed PAECs. The overexpression of CHIP potentiated, whereas a CHIP U-box domain mutant attenuated, ADMA-induced GCH1 degradation and reductions in cellular BH(4) concentrations. We also found in vivo that Hsp90/GCH1 interactions are decreased, whereas GCH1-Hsp70 and GCH1-CHIP interactions and GCH1 ubiquitination are increased. Finally, we found that supplementation with l-arginine restored Hsp90-GCH1 interactions and increased both BH(4) and NO(x) concentrations in Shunt lambs. In conclusion, increased concentrations of ADMA can indirectly alter NO signaling through decreased cellular BH(4) concentrations, secondary to the disruption of Hsp90-GCH1 interactions and the CHIP-dependent proteasomal degradation of GCH1.

Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase.

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.