Caracterització i propietats de dues endo-B-(1,4)-glucanases implicades en l'estovament del fruit de maduixa.

[cat] Un dels enzims que podria tenir una acció important en l'estovament de la maduixa és l'endo-?-(1,4)-glucanasa (EGasa). Donat que aquest estovament produeix pèrdues comercials molt importants, es va plantejar l'objectiu de caracteritzar la funció biològica de dues endo-?-(1,4)-glucanases aïllades prèviament en fruit de maduixa: Cel1 i Cel2. En maduixa, l'activitat EGasa augmenta durant la maduració, i aquest augment coincideix temporalment amb l'increment d'expressió i acumulació proteica de Cel1 i Cel2. L'expressió superposada de Cel1 i Cel2 en maduixa observada per Northern-blot, podria indicar una certa cooperació d'aquests dos enzims en el metabolisme dels polímers de la paret cel·lular que acompanya a la maduració del fruit. Cel1 en maduixera no s'expressa en teixits diferents del fruit, per tant, es pot afirmar que entre totes les EGases descrites fins ara, i que s'expressen en fruits, Cel1 és la que té el patró d'expressió més específic de maduració. L'acumulació de Cel2 durant el procés de maduració suggereix un paper important d'aquesta EGasa en l'estovament dels fruits. A més, l'expressió de Cel2 en teixits en creixement dóna suport a una participació d'aquest gen en les modificacions de la paret cel·lular que acompanyen el creixement i l'expansió cel·lular. El punt isoelèctric trobat per Cel1 és de 8'5, molt proper al valor teòric predit a partir de la seqüència. Cel2 en canvi, presenta un punt isoelèctric de 5'0, molt diferent del valor teòric predit. Ambdues proteïnes presenten un senyal consens per la N-glicosilació, però només Cel1 té afinitat per la concanavalina A. Per tant, o bé només Cel1 està realment glicosilada in vivo, o més probablement, Cel1 i Cel2 es conjuguen amb residus glucosídics diferents. L'eliminació dels aquenis del fruit inicia el procés de maduració de la maduixa, provocant un augment de l'expressió i de l'acumulació proteica de Cel1 i Cel2. Donat que en els aquenis es produeix la síntesi i alliberació d'auxines, es pot afirmar que Cel1 i Cel2 estan sota control negatiu de les auxines. L'aplicació exògena d'etilè en fruits de maduixa no modifica l'expressió de Cel1 i Cel2, indicant que aquesta hormona no intervé en la regulació transcripcional d'aquests gens. S'ha detectat cinc isoformes amb activitat endoglucanasa en extractes proteics totals de maduixa madura; una isoforma bàsica i quatre isoformes àcides. Mitjançant Western-blot del gel nadiu, s'ha vist que la isoforma de pI bàsic (pI 8'5) es correspon a Cel1, mentre una de les isoformes àcides, de pI 5-5'5, es correspon a la proteïna Cel2. La sobreexpressió de Cel1 en el llevat Pichia pastoris produeix una proteïna que es troba glicosilada i probablement unida iònicament a la paret cel·lular del llevat. Aquesta proteïna té un pH òptim d'activitat endoglucanasa de 7'5 i ha mostrat afinitat significativa només per substrats amb enllaços ?-(1,4): CMC, xiloglucans i Cel·lulosa CF-11. Per tant, els substrats potencials in vivo per la proteïna Cel1 podrien ser els xiloglucans i/o la cel·lulosa de la paret cel·lular. La inhibició antisentit de l'expressió de Cel1 en plantes transgèniques de maduixa disminueix clarament l'acumulació de la proteïna i resulta en una reducció parcial de l'activitat endoglucanasa total en fruit madur. Estudis preliminars mostren que això es tradueix només en una lleugera disminució de la textura del fruit. Aquests resultats suggereixen que hi ha altres enzims implicats en l'estovament del fruit que compensen la inhibició de Cel1, o bé que la contribució de Cel1 a l'activitat endoglucanasa total és petita. No s'han pogut obtenir plantes de maduixa transgèniques antisentit amb expressió reduïda per Cel2. Aquest fet es podria explicar perquè Cel2 és un gen imprescindible per la planta, i per tant, sense la seva expressió les plantes no es poden desenvolupar

Inflammation and metabolic dysregulation in diabetic cardiomyopathy

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/67430Diabetic cardiomyopathy is characterized by structural and functional alterations in the heart muscle of people with diabetes that finally lead to heart failure. Metabolic disturbances characterized by increased lipid oxidation, intramyocardial triglyceride accumulation and reduced glucose utilization have all been involved in the pathogenesis of diabetic cardiomyopathy. On the other hand, evidences arisen in the recent years point to a potential link between chronic low-grade inflammation in the heart and metabolic dysregulation. Interestingly, the progression of heart failure and cardiac hypertrophy usually entails the activation of pro-inflammatory pathways. Therefore, in this chapter we summarize novel insights into the crosstalk between inflammatory processes and metabolic dysregulation in the failing heart during diabetes

Emerging Actors in Diabetic Cardiomyopathy: Heartbreaker Biomarkers or Therapeutic Targets?

The diabetic heart is characterized by metabolic disturbances that are often accompanied by local inflammation, oxidative stress, myocardial fibrosis, and cardiomyocyte apoptosis. Overall changes result in contractile dysfunction, concentric left ventricular (LV) hypertrophy, and dilated cardiomyopathy, that together affect cardiac output and eventually lead to heart failure, the foremost cause of death in diabetic patients. There are currently several validated biomarkers for the diagnosis and risk assessment of cardiac diseases, but none is capable of discriminating patients with diabetic cardiomyopathy (DCM). In this review we point to several novel candidate biomarkers from new activated molecular pathways (including microRNAs) with the potential to detect or prevent DCM in its early stages, or even to treat it once established. The prospective use of selected biomarkers that integrate inflammation, oxidative stress, fibrosis, and metabolic dysregulation is widely discussed

The PPARβ/δ-AMPK Connection in the Treatment of Insulin Resistance

The current treatment options for type 2 diabetes mellitus do not adequately control the disease in many patients. Consequently, there is a need for new drugs to prevent and treat type 2 diabetes mellitus. Among the new potential pharmacological strategies, activators of peroxisome proliferator-activated receptor (PPAR)β/δ show promise. Remarkably, most of the antidiabetic effects of PPARβ/δ agonists involve AMP-activated protein kinase (AMPK) activation. This review summarizes the recent mechanistic insights into the antidiabetic effects of the PPARβ/δ-AMPK pathway, including the upregulation of glucose uptake, muscle remodeling, enhanced fatty acid oxidation, and autophagy, as well as the inhibition of endoplasmic reticulum stress and inflammation. A better understanding of the mechanisms underlying the effects resulting from the PPARβ/δ-AMPK pathway may provide the basis for the development of new therapies in the prevention and treatment of insulin resistance and type 2 diabetes mellitus

Endoplasmic reticulum stress downregulates PGC-1α in skeletal muscle through ATF4 and an mTOR-mediated reduction of CRTC2

Background Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) downregulation in skeletal muscle contributes to insulin resistance and type 2 diabetes mellitus. Here, we examined the effects of endoplasmic reticulum (ER) stress on PGC-1α levels in muscle and the potential mechanisms involved. Methods The human skeletal muscle cell line LHCN-M2 and mice exposed to different inducers of ER stress were used. Results Palmitate- or tunicamycin-induced ER stress resulted in PGC-1α downregulation and enhanced expression of activating transcription factor 4 (ATF4) in human myotubes and mouse skeletal muscle. Overexpression of ATF4 decreased basal PCG-1α expression, whereas ATF4 knockdown abrogated the reduction of PCG-1α caused by tunicamycin in myotubes. ER stress induction also activated mammalian target of rapamycin (mTOR) in myotubes and reduced the nuclear levels of cAMP response element-binding protein (CREB)-regulated transcription co-activator 2 (CRTC2), a positive modulator of PGC-1α transcription. The mTOR inhibitor torin 1 restored PCG-1α and CRTC2 protein levels. Moreover, siRNA against S6 kinase, an mTORC1 downstream target, prevented the reduction in the expression of CRTC2 and PGC-1α caused by the ER stressor tunicamycin. Conclusions Collectively, these findings demonstrate that ATF4 and the mTOR-CRTC2 axis regulates PGC-1α transcription under ER stress conditions in skeletal muscle, suggesting that its inhibition might be a therapeutic target for insulin resistant states

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription

SIRT3 deficiency exacerbates fatty liver by attenuating the HIF1α-LIPIN 1 pathway and increasing CD36 through Nrf2

Background: Deficiency of mitochondrial sirtuin 3 (SIRT3), a NAD+ -dependent protein deacetylase that maintains redox status and lipid homeostasis, contributes to hepatic steatosis. In this study, we investigated additional mechanisms that might play a role in aggravating hepatic steatosis in Sirt3-deficient mice fed a high-fat diet (HFD). Methods: Studies were conducted in wild-type (WT) and Sirt3−/− mice fed a standard diet or a HFD and in SIRT3- knockdown human Huh-7 hepatoma cells. Results: Sirt3−/− mice fed a HFD presented exacerbated hepatic steatosis that was accompanied by decreased expression and DNA-binding activity of peroxisome proliferator-activated receptor (PPAR) α and of several of its target genes involved in fatty acid oxidation, compared to WT mice fed the HFD. Interestingly, Sirt3 deficiency in liver and its knockdown in Huh-7 cells resulted in upregulation of the nuclear levels of LIPIN1, a PPARα co-activator, and of the protein that controls its levels and localization, hypoxia-inducible factor 1α (HIF-1α). These changes were prevented by lipid exposure through a mechanism that might involve a decrease in succinate levels. Finally, Sirt3−/− mice fed the HFD showed increased levels of some proteins involved in lipid uptake, such as CD36 and the VLDL receptor. The upregulation in CD36 was confirmed in Huh-7 cells treated with a SIRT3 inhibitor or transfected with SIRT3 siRNA and incubated with palmitate, an effect that was prevented by the Nrf2 inhibitor ML385. Conclusion: These findings demonstrate new mechanisms by which Sirt3 deficiency contributes to hepatic steatosi

Design and Synthesis of AMPK Activators and GDF15 Inducers

Targeting growth differentiation factor 15 (GDF15) is a recent strategy for the treatment of obesity and type 2 diabetes mellitus (T2DM). Here, we designed, synthesized, and pharmacologically evaluated in vitro a novel series of AMPK activators to upregulate GDF15 levels. These compounds were structurally based on the (1-dibenzylamino-3-phenoxy)propan-2-ol structure of the orphan ubiquitin E3 ligase subunit protein Fbxo48 inhibitor, BC1618. This molecule showed a better potency than metformin, increasing GDF15 mRNA levels in human Huh-7 hepatic cells. Based on BC1618, structural modifications have been performed to create a collection of diversely substituted new molecules. Of the thirty-five new compounds evaluated, compound 21 showed a higher increase in GDF15 mRNA levels compared with BC1618. Metformin, BC1618, and compound 21 increased phosphorylated AMPK, but only 21 increased GDF15 protein levels. Overall, these findings indicate that 21 has a unique capacity to increase GDF15 protein levels in human hepatic cells compared with metformin and BC1618

PPARβ/δ: A Key Therapeutic Target in Metabolic Disorders

Research in recent years on peroxisome proliferator-activated receptor (PPAR)β/δ indicates that it plays a key role in the maintenance of energy homeostasis, both at the cellular level and within the organism as a whole. PPARβ/δ activation might help prevent the development of metabolic disorders, including obesity, dyslipidaemia, type 2 diabetes mellitus and non-alcoholic fatty liver disease. This review highlights research findings on the PPARβ/δ regulation of energy metabolism and the development of diseases related to altered cellular and body metabolism. It also describes the potential of the pharmacological activation of PPARβ/δ as a treatment for human metabolic disorder

Oral administration of a new HRI activator as a new strategy to improve high-fat-died-induced glucose intolerance, hepatic steatosis and hypertriglyceridemia through FGF21

BACKGROUND AND PURPOSE Fibroblast growth factor 21 (FGF21) has emerged as a therapeutic strategy for treating type 2 diabetes mellitus due to its antidiabetic effects, and this has led to the development of FGF21 longacting analogs. These compounds have some limitations, including requiring subcutaneous injection and their prolonged pharmacodynamic effect compared with native FGF21, which might be responsible for their reported side effects. EXPERIMENTAL APPROACH We have previously demonstrated that intraperitoneal administration of heme-regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI) activators increases hepatic and circulating levels of FGF21. In this study, we examined the effects of oral administration of a new HRI activator, EPB-53, on high-fat diet (HFD)-induced glucose intolerance, hepatic steatosis, and hypertriglyceridemia, compared with metformin. KEY RESULTS Administration of EPB-53 administration for the last two weeks, to mice fed a HFD for 10 weeks, reduced body weight gain, improved glucose intolerance, and prevented hepatic steatosis and hypertriglyceridemia; whereas metformin only ameliorated glucose intolerance. Moreover, EPB- 53, similarly to the reported effects of FGF21, reduced lipogenesis in cultured human hepatocytes and in the liver of mice fed a HFD. Administration of EPB-53 to Fgf21-knockout mice had no effects, demonstrating that its efficacy is dependent on this hormone. CONCLUSIONS AND IMPLICATIONS Overall, the findings of this study demonstrate that oral administration of HRI activators is a promising strategy for the treatment of type 2 diabetes mellitus and non-alcoholic fatty liver disease by increasing FGF21