The 5’-nucleotidases are a family of enzymes that catalyze the dephosphorylation of nucleoside monophosphates and regulate cellular nucleoside and nucleotide levels (Bianchi and Spychala, 2003). The 5’-nucleotidase was first studied in vitro using a semi-purified enzymes extracted from rat and guinea pig skeletalmuscle (Cozzani et al., 1969). There are various biochemical pathways that govern purine and pyrimidine nucleotide metabolism. These pathways maintain the levels of purine and pyrimidine nucleotide triphosphates vital to support various cellular processes. 5’-nucleotidases catabolize nucleoside monophosphates and change their abundance. Therefore they are components of cellular energy homeostasis. Besides maintaining balanced ribo and deoxyribonucleotide pools, nucleotidase activities are likely to regulate the activation of nucleoside analogues, a class of anti-cancer and anti-viral agents that rely on the nucleoside kinases for phosphorylation to convert to their phosphorylated active forms (Hunsucker et al., 2005). Early studies on 5’-nucleotidases have revealed a membrane bound ecto-enzyme and soluble form cytosolic enzymes. In humans, seven 5’- nucleotidases have been isolated and characterized that vary in subcellular localization. Out of the seven known 5’-nucleotidases, five are of the soluble form and they are localized to cytosol, one is localized to the mitochondrial matrix (NT5M) and one is bound to the extracellular portion of the plasma membrane (the ecto 5’-nucleotidase or E5’N) (Hunsucker et al., 2005). These enzymes have similar functions in that they hydrolyze 5’ nucleoside monophosphates, but they differ in their specificity towards their substrate. Some of these enzymes are ubiquitously distributed and some are tissue- specific. Differences in subcellular localization, specificity towards their substrate and tissue specific distribution allow regulation of nucleotide pools to meet the energy balance and cellular homeostasis. This thesis will focus on two of the soluble 5’- nucleotidases enzymes, namely NT5C1A and NT5C2.
Fructose may cause insulin resistance by accumulation of triglycerides in the liver. There are two metabolic pathways to increased hepatic lipid content, i.e., lipogenesis and/or reduced mitochondrial fatty acid oxidation. Hepatic fructolysis leads to increased gluconeogenic sources resulting in elevated rates of lipogenesis [16,45,46]. Hepatic accumulation of toxic intermediary lipid metabolites, such as diacylglycerol (DAG) results in PKC ε activation that impairs hepatic insulin signaling through phosphorylation of serine residues on the insulin receptor substrate 1 and 2 (IRS1/2). When hepatic insulin signaling is impaired, gluconeogenesis and glycogenolysis are unleashed, contributing to hyperglycemia and hyperinsulinemia. Under these circumstances, hepatic lipid synthesis is enhanced due to hyperinsulinemia [241,242]. Likewise, reduced fatty acid oxidation leads to hepatic triglycerides accumulation. Of note, Ohashi et al. demonstrated that excessive amounts of fructose consumption lead to epigenetic modifications, such as DNA hypermethylation of promoter regions of peroxisome proliferator-activated receptor alpha (PPAR α ) and carnitine palmitoyl transferase 1A (CPT1A) that results in lower amounts of mRNA levels . Hepatic triglyceride accumulation results in augmented secretion of very low-density lipoprotein (VLDL) leading to increased lipid uptake inskeletalmuscleand peripheral tissues. Similarly to what happens in the liver, intramyocellular lipid accumulation (particularly DAG) activates the PKC θ isoform that phosphorylates and inactivates IRS1 resulting in impaired insulin-stimulated glucose uptake, contributing to hyperglycemia, increased delivery ofglucose to the liver, and hyperinsulinemia [241,242].
Central ghrelin effects are particularly intriguing in the case of AMPK, CPT1, and malonyl-CoA levels. Preceding studies demonstrated that peripheral and central administration of ghrelin to rats affects AMPK activity in a tissue-specific manner. AMPKα is activated in the brain and heart, whereas it is inhibited in liver and adipose tissue, and no effect is detected on skeletalmuscle (17, 54, 60–63). Our data show for first time that chronic ghrelin treatment enhanced protein levels of AMPKα and pAMPKα. ACC activity was enhanced after central ghrelin infusion in liver and WAT, but the levels of its product, malonyl-CoA, were decreased in both tissues of wild-type Lewis rats. A reasonable explanation for this is that central ghrelin treatment increased the activities of FAS (liver and WAT) and MCD (only in WAT), leading to an increase of malonyl-CoA turnover. Malonyl-CoA acts as negative mediator of fatty acid oxidation by inhibiting CPT-1 and blocking entry of fatty acids into the mitochondria for β-oxidation (64). Interestingly, our results suggest that hepatic CPT1 is regulated in a GH-dependent manner because we observed that chronic infusion of ghrelin directly into the CNS decreased protein and activity levels of CPT1 only in the liver of wild-type Lewis rats and not in dwarf rats. This result suggests that the potential of central ghrelin to promote hepatic lipids storage is higher in a GH-dependent- (favoring lipid deposition and decreasing lipid mobilization) than in a GH-independent manner (favoring only lipid deposition). Contrary to what happens in liver, central ghrelin infusion increased CPT1 protein and activity levels in WAT, independent of GH levels. Nevertheless, activation of the central ghrelin system may increase lipid oxidation in WAT, and our data indicate that fat mass and fat storage enzymes were also stimulated by ghrelin. Thereby, our data suggest that the enhanced β-oxidation in WAT after central ghrelin infusion might be a compensatory mechanism and is a GH-independent effect.
large capacity to store triglycerides during feeding, as well as to hydrolyse and release triglycerides as FFAs and glycerol during fasting. Apart from their storage function, adipocytes secrete a large number of hormones and cytokines (known as adipokines) that affect energy metabolismin other tissues (Guilherme et al., 2008). As overfeeding develops, adipocytes enlarge as a result of increased triglyceride deposition. This enlargement rises the rates of lipolysis (Arner, 2005), consequently increasing the levels of circulating FFA, and also promotes the secretion of inflammatory cytokines. The action of such cytokines profoundly affects the adipocyte function by further increasing lipolysis and inhibiting TG synthesis (Guilherme et al., 2008). The release of FFA as a result of increased adipose lipolysis, may be the single most critical factor in modulating insulin sensitivity in peripheral tissues (Kahn et al., 2006). The excessive circulating FFAs cause accumulation of triglycerides into non- adipose tissues, such as liver andskeletalmuscle, which contribute to the development of insulin resistance in these tissues. (Krssak et al., 1999; Perseghin et al., 1999). Specifically, FFA would promote insulin resistance by inhibiting glucose oxidation (Randle cycle)(Bevilacqua et al., 1990). Additionally, the cytosolic accumulation of triglycerides and derived lipid intermediates, such as ceramides and diacylglycerol (DAG), interfere with the insulin signalling pathway in these tissues, thus promoting insulin resistance. (Muoio et al., 2008). Along with the developing hyperglycaemia resulting from the insulin resistance in peripheral tissues, a chronic elevation in FFA impairs the β-cell secretory function and induces β-cell apoptosis, thus possibly contributing to the β-cell failure and reduced β-cell mass observed in the progression to T2DM (Poitout et al., 2008).
Bile acids, known mainly for their participation in the absorption of lipids and fat-soluble vitamins, have also an important role in the metabolismoflipid, glucose, and en- ergy expenditure. FXR and TGR5/M-BAR, two signaling pathways, are important bile acid synthesis regulators. Also, it has been revealed that they are relevant metabolic regulators for maintaining glucose homeostasis and has converted them in possible new therapeutic targets for dia- betes. Nowadays, the only approved therapeutic option for the treatment of diabetes, related to BAs, involves the use of bile acid chelates. However, it has been shown that some of the benefits of bariatric surgery on glucose con- trol in diabetic patients are related to bile acid metabo- lism.
The FA accumulation observed in the Drosophila FRDA model is in good agreement with the lipid inclusions found previously in the cardiac muscleof FRDA conditional mouse models (33) and with the increased intracellular lipid content present in the striated muscle fibers and Schwann cells of inherited a-tocopherol deficiency patients (21), a disease that appears to be clinically indistinguishable from FRDA. Moreover, up to 40% of FRDA patients manifest different glucosemetabolism problems (53) such as diabetes or insulin resistance, and remarkably type 2 diabetes patients also present an altered lipid status attributable to a mitochon- drial dysfunction (54,55). However, the link between increased lipid amounts and FRDA is not completely clear. On the one hand, depletion of frataxin generates different scenarios that can lead to the inhibition of mitochondrial b-oxidation. For example, mitochondrial respiration defects induce a reduction of NAD + /NADH ratios compromising the b-oxidation pathway (22,56). Alternatively, impairment of aconitase activity increases citrate levels (57), and citrate is an allosteric activator of malonyl-CoA production, which in turn is a potent inhibitor of the mitochondrial b-oxidation (reviewed in 58). Furthermore, downregulation of peroxisome proliferator-activated receptor gamma (PPARg) pathway has been observed in FRDA mice models, suggesting a reduction oflipid catabolism (59). Moreover, on the other hand, frataxin deficiency has been suggested to increase lipogenesis via hyperactivation of mitochondrial acyl-CoA thioesterase (60) or upregulating the expression of the sterol-responsive element-binding protein 1 (Srebp1) (59). Thus, derangement oflipid homeostasis in FRDA could be produced by either blocking the main degradation pathway of FAs or increasing their synthesis. In addition, lipid imbalance could be a critical event in FRDA progression since lipidmetabolism is the main fuel source of the cell.
Conventional (c) and novel (n) PKC:s have a negative impact on the insulin signaling pathway. These serine kinases can be activated by DAG and they induce insulin resistance by phosphorylating defined residues of IRS-1 (Yu, Chen et al. 2002; Ragheb, Shanab et al. 2009). Among the several isoforms of novel/conventional PKCs, the PKCθ and PKCε are mainly associated with DAG-induced skeletalmuscle insulin resistance in rodents and humans (Idris, Gray et al. 2001; Samuel, Petersen et al. 2010). Moreover, PKCθ knockout mice are prevented from muscle insulin resistance during lipid infusion (Kim, Fillmore et al. 2004). Increasing DAG content via inhibition of diacylglycerol kinase δ (DGKδ) results in activation of n/c PKCs and increases phosphorylation of IRS-1 at Ser 307 in intact skeletalmuscle from rat (Chibalin, Leng et al. 2008). Besides direct inhibitory phosphorylation of IRS-1, PKCs can act upstream of the stress kinases JNK and IKKβ and thereby influence insulin signaling by serine phosphorylation of IRS-1. PKCε has also been shown to interfere with insulin signaling by directly associating with and inhibiting the tyrosine kinase activity of the insulin receptor inskeletalmuscleof diabetic animals (Ikeda, Olsen et al. 2001). Finally, PKCζ has been shown to mediate ceramide-induced insulin resistance, as discussed in section 220.127.116.11.
The studies of Hill et al. (2014) and Hamlin et al. (2012) reported a reduction in post-exercise muscle pain when using GCGs. This finding can be explained by the improvement in blood circulation, allowing greater efficiency in venous blood removal, as well as reducing muscle microtrauma, promoting reduction of swelling and psychological comfort. In this sense, the benefits of GCGs use seem to be an important aid in recovery (Hamlin et al., 2012; Hill et al., 2014). Since the rules of many competitive sports do not allow the use of GCGs during competition, research has suggested that compression clothes may be suitable as a recovery aid (Kraemer et al., 1996). In this sense, reduction in several blood variables associated with muscle injury andmetabolism were reported after using GCGs as a recovery aid (Goto & Morishima, 2014). However, studies reporting results of performance-related blood markers to support the use of garments in order to improve recovery are scarce (Kraemer et al., 1996).
almost completely abolishing the slow calcium component. A complete blockade of the fast signal was not expected, consid- ering that mechanisms for depolarization-evoked fast calcium signals in myotubes are well known; they depend on RyRs and are related to excitation-contraction coupling (1, 3, 4). In this context, nucleotides could only contribute toward modulation of this pathway. It is worth speculating that ATP extrusion after tetanic stimulation could contribute to supplementing the cal- cium needed to maintain contraction. Excitation-coupled cal- cium entry is a well known process in myotubes, and it has been linked to DHPR activity (66). Although important in myotubes, the contribution of purinergic receptors to the fast calcium transient appears to be less prominent in adult fibers. Only a 20% reduction of this transient was evident in these cells after a 20-min apyrase treatment (Fig. 3E). In contrast, the extracellu- lar nucleotide pathway appears to be essential for the develop- ment of slow calcium signals generated by electrical depolariza- tion inskeletal myotubes and adult fibers. MRS2179, a selective blocker of P2Y 1 receptor subtype, slightly but significantly
Studies on the inflammatory infiltrate: The technique of Maskrey et al. (1977) was used with sorne modifications. Groups of four mice (20-22 g) were injected iro. in the right gastrocnemius with 1 00 J..Lg of venom . At different time intervals (6 hr, 24 hr, 48 hr and 72 hr) mice were killed and envenomated muscle Was removed and chopped with scissors in 2 .0 mI of phosphate-buffered saline. The tissue suspension was then incubated , with continuous agitation , for 30 min at 37 C. The suspension was flltered through bolting silk and inflammatory cells were counted in a hemocy tometer. Just before counting, the cell suspension was treated with a solution (3% acetic acid) that lyses erythrocytes whlle leaving leucocytes and macrophages intact ; this was done in order to count only inflammatory cells and not erythrocytes present in the tissue due to hemorrhage . Then, the suspension Was centrifuged in a cytocentrifuge and smears were prepared and stained with Wright in order to identify the inflammatory cells. Cells were classified as macrophages, polymorphonuclear leucocytes and lymphocytes.
against HCV. Scientifi cally, the absence of a comple- te defi nition of immunologic parameters correlating with protection and/or the clearance of HCV, and particularly the controversial role of neutralizing an- tibodies, are probably the most important elements related to this situation. In favor of antibody respon- se, subjects with primary hypogammaglobulinemia showed rapid disease progression and poor response to interferon treatment . Moreover, previous stu- dies reported the presence of antibodies specifi c to E2 HVR in individuals who spontaneously resolved HCV infection [45, 46]. However, there is relevant data on the null or delayed induction of neutralizing antibodies in HCV infection [47, 48]. Additionally, since at least some neutralizing antibodies are di- rected towards HVR-I, the induction of this type of response has been involved in selecting viral diver- sity and a mechanism for viral escape. In other ca- ses, neutralizing antibodies cross-reacting with HCV isolates from different genotypes have been found in chronically infected HCV patients, indicating a high degree of conservation of the targeted epitope [49, 50]. Nevertheless, these antibodies, even when indu- ced at high levels, are unable to clear chronic HCV infection .
Several anti-fibrotics have been tested to decrease fibro- sis associated to dystrophic skeletalmuscle . Among them neutralizing antibodies against all three forms of TGF-β importantly reduced hydroxyproline levels and plasma creatine kinase, improved respiratory function and grip strength . Halofunginone has been tested in mdx mice, reducing collagen content and improving respiratory and heart function. It has been suggested that it inhibits p-Smad-3 in response to TGF-β1 [45-47]. The use ofin- hibitors and antagonists of the renin-angiotensin system have been shown to decrease fibrosis and improve skeletalmuscle function . Infusion of angiotensin 1-7, which signals through the Mas receptor, has been shown to importantly decrease fibrosis, TGF-β mediated signaling and increase skeletalmuscle strength . It is difficult to compare which of these drugs, including andrographolide, have a better effect on dystrophic skeletalmuscle, since some of them may also have other undesired side ef- fects. Furthermore, the same readouts are not always determined in each case. Nevertheless a comparative study, under the same experimental conditions would be very valuable.
The results of the present study indicate that Met, phospho-Met and glypican-1 colocalized inlipid raft do- mains of the plasma membrane. Moreover, glypican-1 expression andlipid raft integrity were required to sus- tain the HGF-dependent signaling. Next, we evaluated whether glypican-1 per se or its presence inlipid raft do- mains was required to sustain the HGF signaling mediated by the Met receptor. A chimeric form of HSPG containing the extracellular domain of rat glypican-1 and the trans- membrane and cytoplasmic domains of mouse syndecan- 1 (F-GlySyn) was expressed in WT cells. This chimeric form localized in the non-lipid-raft region of the plasma membrane as we previously reported . Figure 5 shows that mock-transfected WT myoblasts induced the activa- tion of AKT and ERK1/2 in response to HGF. In myo- blasts expressing the chimeric F-GlySyn, however, both phospho-AKT and phospho-ERK1/2 levels decreased com- pared to WT cells. These levels are comparable to levels found in the glypican-1-deficient myoblasts. The figure also shows that diminished sensitivity to HGF, which we had previously observed in the glypican-1-deficient cells, was restored after reexpressing glypican-1 by transient transfection with rat glypican-1. Together, these results in- dicate that glypican-1 must be associated with lipid rafts to sustain HGF-dependent signaling.
Patients with LC, who came to our hospital from August 2007 to August 2010, were prospectively evaluated. A cohort of 130 patients was selected ran- domly. The patients were older than 18 years, with LC of diverse etiology. Diagnosis of cirrhosis was made by liver biopsy or a combination of clinical and laboratory data and imaging studies. Patients with clinical complications due to liver disease were eliminated: hepatocellular carcinoma, alcoholic he- patitis, active gastrointestinal bleeding, clinically or ultrasonographically detected ascites, clinically evident hepatic encephalopathy (according with West Haven criteria), hepatorenal syndrome, and se- vere infection. Patients under effective treatment of previously detected ascites, portal hypertension or hepatic encephalopathy were included. In order to avoid confounding results of plasma glucose tests patients with acute and chronic pancreatitis, pan- creatic cancer and pancreatectomy, endocrinopa- thies (such as Cushing syndrome, acromegaly,
Profibrotic factors reside mainly in the interstitial space and are increased in fibrotic skeletalmuscle [6, 15, 16]. It has been reported that transforming growth factor β (TGF-β), connective tissue growth factor (CCN2/CTGF) , platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), among others [2, 18], can interact directly with ECM proteins. In this manner, they create a profibrotic environment that modulates locally different cell types residing inskeletalmuscle tissue to induce ECM accumulation. TGF-β signaling is involved in several physiological processes such as development, migration and ECM production . Moreover, TGF-β overexpression has also been implied in pathologies such as cancer, autoimmune, cardiovascular and fibrotic diseases including muscle pathologies. TGF-β is secreted mainly by macrophages in the skeletalmuscle during early stages of regeneration but remains persistent in fibrotic muscles [6-8, 16, 20-25]. TGF-β signals through the canonical Smad-dependent pathway or Smad-independent pathways [26, 27]. Canonical TGF-β signaling begins with the activation of the TGF-β receptor I kinase followed by heterodimerization with TGF-β receptor II after ligand binding. Subsequent phosphorylation of Smad2/3 allows binding to Smad4 and translocation into the nucleus, where this protein complex drives the expression of target genes such as collagen, fibronectin, and CCN2 among others [27-30]. TGF-β also signals through several non-canonical pathways including MAPKs (ERK, p38, JNK), AKT, JAK1, Smad1 and others, differentially among cell types [31, 32]. CCN2 is a crucial profibrotic factor that is a member of the CCN family of matricellular proteins. It promotes fibroblast proliferation, ECM production, cell adhesion and migration of a variety of cell types including skeletalmuscle cells [2, 30, 33-36]. CCN2 is vital during development , barely expressed in adult normal healthy muscles  and restricted to early steps of wound healing . In contrast, its expression is increased in challenged skeletalmuscle, e.g. in pathological conditions such as dystrophic skeletalmuscle from the mdx mouse model for Duchenne muscular dystrophy (DMD) [15,39, 40], under conditions of repetitive damage , in a transgenic mouse model for Amyotrophic lateral sclerosis (ALS, tg hSOD1G93A) [7, 41], and after denervation by sciatic nerve transection . Accordingly, CCN2 reduction or blockage attenuates skeletalmuscle fibrosis in these models [9, 15, 41,42], demonstrating its critical role in fibrosis progression.
Since glypican-1 localized in raft domains would be responsible for the sequestering of FGF-2, we expressed a chimeric form of a HSPG containing the extracellular domain of rat glypican-1 and the transmembrane and cytoplasmic domains of mouse synde- can-1 containing a FLAG epitope (F-GlySyn). C6 myoblasts were transfected with F-GlySyn, lysed, and subjected to sucrose density fractionation. Figure 8A shows that the chimeric HSPG revealed by an anti-FLAG immunoblot migrated only at high-density frac- tions. The signaling mediated by FGF-2 in C6 myoblasts express- ing F-GlySyn form was evaluated. Figure 8B shows that in F- GlySyn-transfected myoblasts, FGF-2 induces phospho-ERK1/2 to levels even higher than observed with the mock-transfected or glypican-1-rescued C6 myoblasts. Consistently, the induction of myogenin and myosin diminished when the chimeric HSPG form was expressed compared to that seen with control transfected or glypican-1-rescued C6 myoblasts, as shown in Fig. 8C. These results suggest that the F-GlySyn form, present in nonraft do- mains, acts as a presenter of FGF-2 to its transducing receptors. If so, F-GlySyn should interact with the FGFRs. Figure 8D shows that in coimmunoprecipitation experiments with anti-FLAG an- tibodies, FGFR-IV was coimmunoprecipitated with F-GlySyn. As expected, rat glypican-1 containing a FLAG epitope as well as mock myoblasts did not coimmunoprecipitate any FGFR- IV. As a positive control, syndecan-4 was coimmunoprecip- tated with FGFR-IV (Fig. 8D). The same figure shows ex- pression of rat glypican-1 and the chimeric F-GlySyn, as determined by immunoreactivity with the anti glypican-1 antibody. Finally, the presence of syndecan-4 coimmunoprecipi- tated from control C6 myoblasts is shown. The results described above clearly indicate that glypican-1 modulates muscle differen- tiation processes, most likely by sequestering FGF-2 inlipid raft domains, avoiding interaction of the ligand with its receptors.
b -cell mass can be increased by augmented proliferation and/ or decreased b-cell death. To understand how b-cell number was increased in Epoxy treated mice we quantified both. b-cell death was detected by TUNEL staining, and proliferation by measur- ing BrdU incorporation in insulin-positive cells. Our results showed a 50% decrease in b-cell death in Epoxy treated b-cells (Fig. 3A-B), and a 2-fold increase in b-cell proliferation (Fig. 3C-D). Interestingly, this proliferative effect is specific on pancreatic b -cells, since other endocrine cells as a -cells showed similar proliferation in both conditions (0.04 § 0.03 % vs. 0.09 § 0.05 %; p D 0.49). These results support the view that Epoxy is an inhibitor of b -cell apoptosis under STZ toxicity, and it enhances b -cell proliferation in vivo.