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BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that commonly results from a high-calorie diet and sedentary lifestyle, leading to insulin resistance and glucose homeostasis perturbation. Physical activity is recommended as one first-line treatment in T2DM, but it leads to contrasted results. We hypothesized that, instead of applying standard exercise protocols, the prescription of personalized exercise programs specifically designed to reverse the potential metabolic alterations in skeletal muscle could result in better results. METHODS: To test this hypothesis, we drew the metabolic signature of the fast-twitch quadriceps muscle, based on a combined unbiased NMR spectroscopy and RT-qPCR study, in several T2DM mouse models of different genetic background (129S1/SvImJ, C57Bl/6J), sex and aetiology (high-fat diet (HFD) or HFD/Streptozotocin (STZ) induction or transgenic MKR (FVB-Tg Ckm-IGF1R*K1003R)1Dlr/J) mice. Three selected mouse models with unique muscular metabolic signatures were submitted to three different swimming-based programs, designed to address each metabolic specificity. RESULTS: We found that depending on the genetic background, the sex, and the mode of T2DM induction, specific muscular adaptations occurred, including depressed glycolysis associated with elevated PDK4 expression, shift to ß-oxidation, or deregulation of amino-acid homeostasis. Interestingly, dedicated swimming-based exercises designed to restore specific metabolic alterations in muscle were found optimal in improving systemic T2DM hallmarks, including a significant reduction in insulin resistance, the improvement of glucose homeostasis, and a delay in sensorimotor function alterations. CONCLUSION: The muscle metabolism constitutes an important clue for the design of precision exercises with potential clinical implications for T2DM patients.
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Diabetes Mellitus Tipo 2 , Modelos Animales de Enfermedad , Músculo Esquelético , Condicionamiento Físico Animal , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus Tipo 2/genética , Músculo Esquelético/metabolismo , Ratones , Masculino , Femenino , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL , Resistencia a la Insulina , Metaboloma , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Ratones Transgénicos , Metabolómica/métodosRESUMEN
GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.
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Axones , Ganglios Espinales , Receptores de GABA , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Ratones , Axones/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA/genética , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ2/genética , Ratones Noqueados , Ratones Endogámicos C57BL , Células Cultivadas , Células de Schwann/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/citología , Técnicas de Cocultivo , Neuronas/metabolismo , Neuronas/efectos de los fármacosRESUMEN
Cancer was recently annexed to diabetic complications. Furthermore, recent studies suggest that cancer can increase the risk of diabetes. Consequently, diabetes and cancer share many risk factors, but the cellular and molecular pathways correlating diabetes and colon and rectal cancer (CRC) remain far from understood. In this study, we assess the effect of hyperglycemia on cancer cell aggressiveness in human colon epithelial adenocarcinoma cells in vitro and in an experimental animal model of CRC. Our results show that Nox (NADPH oxidase enzyme) 4-induced reactive oxygen species (ROS) production is deregulated in both diabetes and CRC. This is paralleled by inactivation of the AMPK and activation of the mammalian target of rapamycin (mTOR) C1 signaling pathways, resulting in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) accumulation, induction of DNA damage, and exacerbation of cancer cell aggressiveness, thus contributing to the genomic instability and predisposition to increased tumorigenesis in the diabetic milieu. Pharmacologic activation of AMPK, inhibition of mTORC1, or blockade of Nox4 reduce ROS production, restore the homeostatic signaling of 8-oxoguanine DNA glycosylase/8-oxodG, and lessen the progression of CRC malignancy in a diabetic milieu. Taken together, our results identify the AMPK/mTORC1/Nox4 signaling axis as a molecular switch correlating diabetes and CRC. Modulating this pathway may be a strategic target of therapeutic potential aimed at reversing or slowing the progression of CRC in patients with or without diabetes.-Mroueh, F. M., Noureldein, M., Zeidan, Y. H., Boutary, S., Irani, S. A. M., Eid, S., Haddad, M., Barakat, R., Harb, F., Costantine, J., Kanj, R., Sauleau, E.-A., Ouhtit, A., Azar, S. T., Eid, A. H., Eid, A. A. Unmasking the interplay between mTOR and Nox4: novel insights into the mechanism connecting diabetes and cancer.
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NADPH Oxidasa 4/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Animales , Antibióticos Antineoplásicos/farmacología , Glucemia , Células CACO-2 , Daño del ADN , Diabetes Mellitus Experimental , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Hipoglucemiantes/farmacología , Masculino , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADPH Oxidasa 4/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Regulación hacia ArribaRESUMEN
The aim of the present study is to take advantage of the unique property of polyisoprenoid chains to adopt a compact molecular conformation and to use these natural and biocompatible lipids as nanocarriers of drugs to deliver siRNA. A new chemical strategy is applied here to conjugate squalene (SQ) and solanesol (SOLA) to siRNA consisting of an activated variant of the azide-alkyne Huisgen cycloaddition also known as copper-free (Cu-free) click chemistry. We conjugated siRNA against TMPRSS2-ERG, a fusion oncogene found in more than 50% of prostate cancers to SQ or SOLA. First, several parameters such as molar ratio, solvents, temperature, incubation time, and the annealing schedule between both siRNA strands were investigated to bioconjugate the SQ or SOLA via Cu-free click chemistry. The best parameters of the new bioconjugation approach allowed us to (i) increase the synthesis yield up to 95%, (ii) avoid the formation of byproducts during the synthesis, and (iii) improve the reproducibility of the bioconjugation. Then, the biological activity of the resulting nanoparticles was assessed. In vitro, all four formulations were able to decrease the corresponding oncogene and oncoprotein expression. In vivo, only two of the four nanoformulations showed anti-neoplastic activity that seems to be tightly related to their dissimilar biodistribution behavior. In conclusion, we performed a new approach easily transposable for pharmaceutical development to synthesize siRNA-SQ and siRNA-SOLA and to obtain efficient siRNA-nanoparticles. The robustness of the process could be extended to several other polyterpenes and likely applied to other siRNA targeting genes whose overexpression results in the development of cancers or other genetic diseases.
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Química Clic , Neoplasias/terapia , Oligonucleótidos/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Tratamiento con ARN de Interferencia , Alquinos/química , Animales , Azidas/química , Línea Celular Tumoral , Química Clic/métodos , Reacción de Cicloadición/métodos , Humanos , Ratones SCID , Nanopartículas/administración & dosificación , Nanopartículas/química , Nanopartículas/uso terapéutico , Neoplasias/genética , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/uso terapéutico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Tratamiento con ARN de Interferencia/métodos , Escualeno/química , Terpenos/químicaRESUMEN
This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/regulation of endothelin-1 (ET-1) to its receptor subtypes A (ETAR) and B (ETBR) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [(125)I]-ET-1 was employed to estimate ET-1 binding affinity (τ = 1/K-n) to ETAR and ETBR at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of ETAR and ETBR were detected using Western blot. No significance differences were detected in the τ of ETAR and ETBR between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the τ value for ETAR and ETBR on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, ETAR and ETBR densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated ETAR and down-regulated ETBR band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus.
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Amidas/farmacología , Vasos Coronarios/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Endotelina-1/metabolismo , Fumaratos/farmacología , Hipoglucemiantes/farmacología , Insulina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Péptidos/farmacología , Receptor de Endotelina A/efectos de los fármacos , Receptor de Endotelina B/efectos de los fármacos , Ponzoñas/farmacología , Animales , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Cardiomegalia/prevención & control , Vasos Coronarios/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Exenatida , Masculino , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Estreptozocina , Factores de TiempoRESUMEN
Small interfering RNAs (siRNA) are attractive and powerful tools to inhibit the expression of a targeted gene. However, their extreme hydrophilicities combined with a negative charge and short plasma half-life counteract their use as therapeutics. Previously, we chemically linked siRNA to squalene (SQ) which self-assembled as nanoparticles (NPs) with pharmacological efficiency in cancers and recently in a hereditary neuropathy. In order to understand the siRNA-SQ NP assembly and fate once intravenously injected, the present study detailed characterization of siRNA-SQ NP structure and its interaction with serum components. From SAXS and SANS analysis, we propose that the siRNA-SQ bioconjugate self-assembled as 11-nm diameter supramolecular assemblies, which are connected one to another to form spherical nanoparticles of around 130-nm diameter. The siRNA-SQ NPs were stable in biological media and interacted with serum components, notably with albumin and LDL. The high specificity of siRNA to decrease or normalize gene expression and the high colloidal stability when encapsulated into squalene nanoparticles offer promising targeted therapy with wide applications for pathologies with gene expression dysregulation.
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Nanopartículas , ARN Interferente Pequeño , Dispersión del Ángulo Pequeño , Escualeno , Difracción de Rayos XRESUMEN
Charcot-Marie-Tooth (CMT) disease is the most frequent inherited neuropathy, affecting 1/1500 to 1/10000. CMT1A represents 60%-70% of all CMT and is caused by a duplication on chromosome 17p11.2 leading to an overexpression of the Peripheral Myelin Protein 22 (PMP22). PMP22 gene is under tight regulation and small changes in its expression influences myelination and affect motor and sensory functions. To date, CMT1A treatment is symptomatic and classic pharmacological options have been disappointing. Here, we review the past, present, and future treatment options for CMT1A, with a special emphasis on the highly promising potential of PMP22-targeted small interfering RNA and antisense oligonucleotides.
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Enfermedad de Charcot-Marie-Tooth/genética , Duplicación de Gen , Proteínas de la Mielina/genética , HumanosRESUMEN
Charcot-Marie-Tooth disease type 1 A (CMT1A) lacks an effective treatment. We provide a therapy for CMT1A, based on siRNA conjugated to squalene nanoparticles (siRNA PMP22-SQ NPs). Their administration resulted in normalization of Pmp22 protein levels, restored locomotor activity and electrophysiological parameters in two transgenic CMT1A mouse models with different severity of the disease. Pathological studies demonstrated the regeneration of myelinated axons and myelin compaction, one major step in restoring function of myelin sheaths. The normalization of sciatic nerve Krox20, Sox10 and neurofilament levels reflected the regeneration of both myelin and axons. Importantly, the positive effects of siRNA PMP22-SQ NPs lasted for three weeks, and their renewed administration resulted in full functional recovery. Beyond CMT1A, our findings can be considered as a potent therapeutic strategy for inherited peripheral neuropathies. They provide the proof of concept for a new precision medicine based on the normalization of disease gene expression by siRNA.
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Enfermedad de Charcot-Marie-Tooth/terapia , Técnicas de Transferencia de Gen , Proteínas de la Mielina/genética , Nanoconjugados , Fibras Nerviosas Mielínicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Tratamiento con ARN de Interferencia , Escualeno/química , Animales , Línea Celular , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Modelos Animales de Enfermedad , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora , Proteínas de la Mielina/metabolismo , Fibras Nerviosas Mielínicas/patología , Regeneración Nerviosa , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , ARN Interferente Pequeño/metabolismo , Recuperación de la Función , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Factores de TiempoRESUMEN
Diabetes triggers peripheral nerve alterations at a structural and functional level, collectively referred to as diabetic peripheral neuropathy (DPN). This work highlights the role of the liver X receptor (LXR) signaling pathway and the cross talk with the reactive oxygen species (ROS)-producing enzyme NADPH oxidase-4 (Nox4) in the pathogenesis of DPN. Using type 1 diabetic (T1DM) mouse models together with cultured Schwann cells (SCs) and skin biopsies from patients with type 2 diabetes (T2DM), we revealed the implication of LXR and Nox4 in the pathophysiology of DPN. T1DM animals exhibit neurophysiological defects and sensorimotor abnormalities paralleled by defective peripheral myelin gene expression. These alterations were concomitant with a significant reduction in LXR expression and increase in Nox4 expression and activity in SCs and peripheral nerves, which were further verified in skin biopsies of patients with T2DM. Moreover, targeted activation of LXR or specific inhibition of Nox4 in vivo and in vitro to attenuate diabetes-induced ROS production in SCs and peripheral nerves reverses functional alteration of the peripheral nerves and restores the homeostatic profiles of MPZ and PMP22. Taken together, our findings are the first to identify novel, key mediators in the pathogenesis of DPN and suggest that targeting LXR/Nox4 axis is a promising therapeutic approach.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Neuropatías Diabéticas/metabolismo , Receptores X del Hígado/metabolismo , NADPH Oxidasa 4/metabolismo , Células de Schwann/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Neuropatías Diabéticas/etiología , Femenino , Humanos , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado/agonistas , Masculino , Ratones , Proteínas de la Mielina/genética , NADPH Oxidasa 4/antagonistas & inhibidores , Pirazoles/farmacología , Pirazolonas , Piridinas/farmacología , Piridonas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Sulfonamidas/farmacologíaRESUMEN
Adhesions after abdomino-pelvic surgery are a cause of morbidity and reoperations. The use of human amniotic membrane (HAM) for adhesion prevention has given controversial results. The mode of administration of the amniotic membrane has not been well studied. This study assessed the efficacy of two modes of application of cryopreserved HAM, patch or fragmented in Lactated Ringer (LR) solution, for the prevention of pelvic adhesion formation postabdomino-pelvic surgery in a mice model. After a midline laparotomy incision, a small cautery lesion was done on each side of the abdominal wall peritoneum in mice. In Group A (control; n = 42), the abdomen was closed directly, Group B (n = 42) received 2.5 ml of LR prior to closure. In Groups C (n = 42) and D (n = 42), a 2 cm × 2 cm patch of HAM and another one fragmented and dispersed in 2.5 ml of LR were applied prior to closure, respectively. Two weeks later, a laparotomy was performed, and gross and pathological evaluation of adhesions, fibrosis, angiogenesis, and inflammation were conducted. Group D exhibited a significantly lower rate of gross adhesion formation. Fibrosis was significantly lowest in Group C as compared to the control. Group B had the lowest vascular formation in the adhesions. The use of HAM fragmented in LR solution is associated with a significantly lower incidence of postoperative adhesions in mice when compared to LR alone, HAM patch, or control. The mechanism of action of this reduction needs to be elucidated by future studies.
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AIM: Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. We explored the hypothesis that mammalian target of rapamycin complex 2 (mTORC2) mediates podocyte injury in diabetes. RESULTS: High glucose (HG)-induced podocyte injury reflected by alterations in the slit diaphragm protein podocin and podocyte depletion/apoptosis. This was paralleled by activation of the Rictor/mTORC2/Akt pathway. HG also increased the levels of Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using small interfering RNA (siRNA)-targeting Rictor in vitro decreased HG-induced Nox1 and Nox4, NADPH oxidase activity, restored podocin levels, and reduced podocyte depletion/apoptosis. Inhibition of mTORC2 had no effect on mammalian target of rapamycin complex 1 (mTORC1) activation, described by our group to be increased in diabetes, suggesting that the mTORC2 activation by HG could mediate podocyte injury independently of mTORC1. In isolated glomeruli of OVE26 mice, there was a similar activation of the Rictor/mTORC2/Akt signaling pathway with increase in Nox4 and NADPH oxidase activity. Inhibition of mTORC2 using antisense oligonucleotides targeting Rictor restored podocin levels, reduced podocyte depletion/apoptosis, and attenuated glomerular injury and albuminuria. INNOVATION: Our data provide evidence for a novel function of mTORC2 in NADPH oxidase-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. CONCLUSION: mTORC2 and/or NADPH oxidase inhibition may represent a therapeutic modality for diabetic kidney disease. Antioxid. Redox Signal. 25, 703-719.
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Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 1/complicaciones , Glucosa/farmacología , Complejos Multiproteicos/metabolismo , NADPH Oxidasas/genética , Podocitos/citología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Apoptosis , Proteínas Portadoras/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteínas de la Membrana/metabolismo , Ratones , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
This study focuses on the effects of long-term renin-angiotensin system suppression and/or incretin mimetic therapies on the regulation and binding affinity of GLP-1 to its receptor in the coronary endothelium (CE) and cardiomyocytes (CMs) of type 1 diabetic male Sprague-Dawley rats. The groups assessed are normal (N), streptozotocin-induced diabetic (D), Insulin treated (DI), Exendin-4 treated (DE), Aliskiren treated (DA), cotreated with Insulin and Aliskiren (DIA) and cotreated with exendin-4 and Aliskiren (DEA). Heart perfusion with (125)I-GLP-1 was performed to estimate GLP-1 binding affinity (τ = 1/k-n) to its receptor in the heart. Western Blotting was assessed to determine the expression variation of GLP-1 receptor in the heart. Plasma GLP-1 levels were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Diabetes decreased the τ value on CE and increased it on CMs compared to normal. The combination of Exendin-4 with Aliskiren showed a normalizing effect on the binding affinity of GLP-1 at the coronary endothelium, while at the cardiomyocyte level Exendin-4 treatment alone was the most effective.