Martin M. Edreira

Skeletal Muscle Triglycerides, Diacylglycerols, and Ceramides in Insulin Resistance: Another Paradox in Endurance-Trained Athletes?

OBJECTIVE Chronic exercise and obesity both increase intramyocellular triglycerides (IMTGs) despite having opposing effects on insulin sensitivity. We hypothesized that chronically exercise-trained muscle would be characterized by lower skeletal muscle diacylglycerols (DAGs) and ceramides despite higher IMTGs and would account for its higher insulin sensitivity. We also hypothesized that the expression of key skeletal muscle proteins involved in lipid droplet hydrolysis, DAG formation, and fatty-acid partitioning and oxidation would be associated with the lipotoxic phenotype. RESEARCH DESIGN AND METHODS A total of 14 normal-weight, endurance-trained athletes (NWA group) and 7 normal-weight sedentary (NWS group) and 21 obese sedentary (OBS group) volunteers were studied. Insulin sensitivity was assessed by glucose clamps. IMTGs, DAGs, ceramides, and protein expression were measured in muscle biopsies. RESULTS DAG content in the NWA group was approximately twofold higher than in the OBS group and ~50% higher than in the NWS group, corresponding to higher insulin sensitivity. While certain DAG moieties clearly were associated with better insulin sensitivity, other species were not. Ceramide content was higher in insulin-resistant obese muscle. The expression of OXPAT/perilipin-5, adipose triglyceride lipase, and stearoyl-CoA desaturase protein was higher in the NWA group, corresponding to a higher mitochondrial content, proportion of type 1 myocytes, IMTGs, DAGs, and insulin sensitivity. CONCLUSIONS Total myocellular DAGs were markedly higher in highly trained athletes, corresponding with higher insulin sensitivity, and suggest a more complex role for DAGs in insulin action. Our data also provide additional evidence in humans linking ceramides to insulin resistance. Finally, this study provides novel evidence supporting a role for specific skeletal muscle proteins involved in intramyocellular lipids, mitochondrial oxidative capacity, and insulin resistance.

Nitro–Fatty Acids Reduce Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Inflammatory processes and foam cell formation are key determinants in the initiation and progression of atherosclerosis. Electrophilic nitro–fatty acids, byproducts of nitric oxide- and nitrite-dependent redox reactions of unsaturated fatty acids, exhibit antiinflammatory signaling actions in inflammatory and vascular cell model systems. The in vivo action of nitro–fatty acids in chronic inflammatory processes such as atherosclerosis remains to be elucidated. Methods and Results—Herein, we demonstrate that subcutaneously administered 9- and 10-nitro-octadecenoic acid (nitro-oleic acid) potently reduced atherosclerotic lesion formation in apolipoprotein E–deficient mice. Nitro–fatty acids did not modulate serum lipoprotein profiles. Immunostaining and gene expression analyses revealed that nitro-oleic acid attenuated lesion formation by suppressing tissue oxidant generation, inhibiting adhesion molecule expression, and decreasing vessel wall infiltration of inflammatory cells. In addition, nitro-oleic acid reduced foam cell formation by attenuating oxidized low-density lipoprotein–induced phosphorylation of signal transducer and activator of transcription-1, a transcription factor linked to foam cell formation in atherosclerotic plaques. Atherosclerotic lesions of nitro-oleic acid-treated animals also showed an increased content of collagen and α-smooth muscle actin, suggesting conferral of higher plaque stability. Conclusion—These results reveal the antiatherogenic actions of electrophilic nitro–fatty acids in a murine model of atherosclerosis.

Downregulation of Rap1GAP through Epigenetic Silencing and Loss of Heterozygosity Promotes Invasion and Progression of Thyroid Tumors

Thyroid cancer is the most common type of endocrine malignancy, encompassing tumors with various levels of invasive growth and aggressiveness. Rap1GAP, a Rap1 GTPase-activating protein, inhibits the RAS superfamily protein Rap1 by facilitating hydrolysis of GTP to GDP. In this study, we analyzed 197 thyroid tumor samples and showed that Rap1GAP was frequently lost or downregulated in various types of tumors, particularly in the most invasive and aggressive forms of thyroid cancer. The downregulation was due to promoter hypermethylation and/or loss of heterozygosity, found in the majority of thyroid tumors. Treatment with demethylating agent 5-aza-deoxycytidine and/or histone deacetylation inhibitor trichostatin A induced gene reexpression in thyroid cells. A genetic polymorphism, Y609C, was seen in 7% of thyroid tumors but was not related to gene downregulation. Loss of Rap1GAP expression correlated with tumor invasiveness but not with specific mutations activating the mitogen-activated protein kinase pathway. Rap1GAP downregulation was required in vitro for cell migration and Matrigel invasion. Recovery of Rap1GAP expression inhibited thyroid cell proliferation and colony formation. Overall, our findings indicate that epigenetic or genetic loss of Rap1GAP is very common in thyroid cancer, where these events are sufficient to promote cell proliferation and invasion.

Phosphorylation-induced Conformational Changes in Rap1b ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser179 at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser179 phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF‐2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1β, TcP2α, TcP2β and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two‐hybrid (Y2H) protein–protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1β–TcP2α and TcP1β–TcP2β. Moreover P2 but not P1 proteins were able to homo‐oligomerize. In addition, the region comprising amino acids 210–270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2β were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF‐2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF‐2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0. Copyright

Identification of novel cyclic nucleotide binding proteins in Trypanosoma cruzi.

Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasites life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known about cAMPs mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A (PKA) has been cloned and characterised in this parasite; however experimental evidence indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new cAMP binding proteins as potential cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a combination of search methods 27 proteins with putative cNMP binding domains (CBDs) were identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, with sequences segregated into two main branches: one containing kinases-like proteins and the other gathering hypothetical proteins with different function or no other known. Comparative modelling of the strongest candidates provides support for the hypothesis that these proteins may give rise to structurally viable cyclic nucleotide binding domains. Pull-down and nucleotide displacement assays strongly suggest that TcCLB.508523.80 could bind cAMP and eventually be a new putative PKA-independent cAMP effector in T. cruzi.

Host Epac1 is required for cAMP-mediated invasion by Trypanosoma cruzi

Mechanistic details of the modulation by cAMP of Trypanosoma cruzi host cell invasion remain ill-defined. Here we report that activation of hosts Epac1 stimulated invasion, whereas specific pharmacological inhibition or maneuvers that alter Epac1 subcellular localization significantly reduced invasion. Furthermore, while specific activation of host cell PKA showed no effect, its inhibition resulted in an increased invasion, revealing a crosstalk between the PKA and Epac signaling pathways during the process of invasion. Therefore, our data suggests that subcellular localization of Epac might be playing an important role during invasion and that specific activation of the host cell cAMP/Epac1 pathway is required for cAMP-mediated invasion.