RESUMEN
Genetically heterogeneous UM-HET3 mice born in 2020 were used to test possible lifespan effects of alpha-ketoglutarate (AKG), 2,4-dinitrophenol (DNP), hydralazine (HYD), nebivolol (NEBI), 16α-hydroxyestriol (OH_Est), and sodium thiosulfate (THIO), and to evaluate the effects of canagliflozin (Cana) when started at 16 months of age. OH_Est produced a 15% increase (p = 0.0001) in median lifespan in males but led to a significant (7%) decline in female lifespan. Cana, started at 16 months, also led to a significant increase (14%, p = 0.004) in males and a significant decline (6%, p = 0.03) in females. Cana given to mice at 6 months led, as in our previous study, to an increase in male lifespan without any change in female lifespan, suggesting that this agent may lead to female-specific late-life harm. We found that blood levels of Cana were approximately 20-fold higher in aged females than in young males, suggesting a possible mechanism for the sex-specific disparities in its effects. NEBI was also found to produce a female-specific decline (4%, p = 0.03) in lifespan. None of the other tested drugs provided a lifespan benefit in either sex. These data bring to 7 the list of ITP-tested drugs that induce at least a 10% lifespan increase in one or both sexes, add a fourth drug with demonstrated mid-life benefits on lifespan, and provide a testable hypothesis that might explain the sexual dimorphism in lifespan effects of the SGLT2 inhibitor Cana.
Asunto(s)
Canagliflozina , Longevidad , Tiosulfatos , Animales , Canagliflozina/farmacología , Masculino , Femenino , Tiosulfatos/farmacología , Longevidad/efectos de los fármacos , Ratones , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Factores SexualesRESUMEN
Mild traumatic brain injury (mTBI) results in impairment of brain metabolism, which is propagated by mitochondrial dysfunction in the brain. Mitochondrial dysfunction has been identified as a pathobiological therapeutic target to quell cellular dyshomeostasis. Further, therapeutic approaches targeting mitochondrial impairments, such as mild mitochondrial uncoupling, have been shown to alleviate behavioral alterations after TBI. To examine how mild mitochondrial uncoupling modulates acute mitochondrial outcomes in a military-relevant model of mTBI, we utilized repeated blast overpressure of 11 psi peak overpressure to model repeated mild blast traumatic brain injury (rmbTBI) in rats followed by assessment of mitochondrial respiration and mitochondrial-related oxidative damage at 2 days post-rmbTBI. Treatment groups were administered 8 or 80 mg/kg MP201, a prodrug of 2,4 dinitrophenol (DNP) that displays improved pharmacokinetics compared with its metabolized form. Synaptic and glia-enriched mitochondria were isolated using fractionated a mitochondrial magnetic separation technique. There was a consistent physiological response, decreased heart rate, following mbTBI among experimental groups. Although there was a lack of injury effect in mitochondrial respiration of glia-enriched mitochondria, there were impairments in mitochondrial respiration in synaptic mitochondria isolated from the prefrontal cortex (PFC) and the amygdala/entorhinal/piriform cortex (AEP) region. Impairments in synaptic mitochondrial respiration were rescued by oral 80 mg/kg MP201 treatment after rmbTBI, which may be facilitated by increases in complex II and complex IV activity. Mitochondrial oxidative damage in glia-enriched mitochondria was increased in the PFC and hippocampus after rmbTBI. MP201 treatment alleviated elevated glia-enriched mitochondrial oxidative damage following rmbTBI. However, there was a lack of injury-associated differences in oxidative damage in synaptic mitochondria. Overall, our report demonstrates that rmbTBI results in mitochondrial impairment diffusely throughout the brain and mild mitochondrial uncoupling can restore mitochondrial bioenergetics and oxidative balance.
Asunto(s)
Traumatismos por Explosión , Conmoción Encefálica , Lesiones Traumáticas del Encéfalo , Profármacos , Ratas , Animales , Profármacos/farmacología , Mitocondrias , Encéfalo , Estrés OxidativoRESUMEN
Dopaminergic neuronal cell loss in the substantia nigra is responsible for the motor symptoms that are the clinical hallmark of Parkinson's disease (PD). As of yet there are no treatments that slow or prevent the degeneration of dopaminergic neurons in PD patients. Here we tested the hypothesis that dopaminergic neurons can be protected by treatment with the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) and the novel DNP prodrug MP201. We found that mice treated with low doses of DNP and MP201 were protected against motor dysfunction and dopamine neuron loss in the 6-hydroxydopamine PD model, with MP201 being more efficacious than DNP. Amelioration of motor deficits and dopamine neuron loss by MP201 treatment was associated with reductions in microglial and astrocyte activation and neuroinflammation. These preclinical findings suggest the potential application of mitochondrial uncoupling agents such as MP201 as disease-modifying therapies for PD.
Asunto(s)
2,4-Dinitrofenol/análogos & derivados , 2,4-Dinitrofenol/uso terapéutico , Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson/tratamiento farmacológico , Profármacos/uso terapéutico , 2,4-Dinitrofenol/farmacología , Animales , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Oxidopamina/farmacología , Enfermedad de Parkinson/patología , Profármacos/farmacologíaRESUMEN
Mitochondrial dysfunction is thought to be involved in the pathogenesis of MS and here we tested if brain penetrant mitochondrial uncouplers, DNP (MP101) and a novel prodrug of DNP (MP201), have the pharmacology to suppress demyelination and axonal loss in two independent models of MS by modulating the entire organelle's physiology. First, the gold standard EAE mouse model for MS was evaluated by daily oral treatment Day 7-21 with either MP101 or MP201 post-immunization. Both MP101/MP201 significantly suppressed progression of paralysis with limited infiltration of inflammatory cells. Strikingly, although mitochondrial uncouplers do increase energy expenditure even at the low doses provided here, they paradoxically preserved body weight at all doses in comparison to wasting in advanced paralysis of the placebos. Second, the effects of the compounds on suppressing inflammation were also evaluated in the cuprizone model, independent of the immune system. MP101/MP201 had a striking effect preserving both myelination and protecting the axons, in comparison to the placebos where both were destroyed. Both MP101/MP201 induced a significant and sustained increase in neurotrophin, BDNF, in the spinal cords. Both MP101/MP201 suppressed the expression of inflammatory cytokines including IL-1ß, TNF-α and iNOS. Results indicate that MP101/MP201 may be a "disease modifying" treatment for MS by specifically modulating mitochondrial physiology. This would be a completely novel treatment for MS, targeting the mitochondria directly using a unique platform, mitochondrial uncouplers, that initially act non-genomically based upon biophysics, but cascades into cellular remodeling, neuroprotection and pro-survival. Clinical Phase I testing of MP101 in Normal Healthy Volunteers (NHV) is currently underway allowing for the potential to subsequently evaluate translation in MS patients and other insidious diseases, at expected weight neutral doses.
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2,4-Dinitrofenol/análogos & derivados , Mitocondrias/efectos de los fármacos , Esclerosis Múltiple/tratamiento farmacológico , Profármacos/uso terapéutico , Desacopladores/uso terapéutico , 2,4-Dinitrofenol/farmacología , 2,4-Dinitrofenol/uso terapéutico , Animales , Axones/efectos de los fármacos , Axones/patología , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Cuprizona , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Preparaciones de Acción Retardada , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/tratamiento farmacológico , Encefalitis/patología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Inmunización , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/patología , Factores de Crecimiento Nervioso/biosíntesis , Parálisis/inducido químicamente , Parálisis/tratamiento farmacológico , Profármacos/farmacologíaRESUMEN
In the sanctity of pure drug discovery, objective reasoning can become clouded when pursuing ideas that appear unorthodox, but are spot on physiologically. To put this into historical perspective, it was an unorthodox idea in the 1950's to suggest that warfarin, a rat poison, could be repositioned into a breakthrough drug in humans to protect against strokes as a blood thinner. Yet it was approved in 1954 as Coumadin® and has been prescribed to billions of patients as a standard of care. Similarly, no one can forget the horrific effects of thalidomide, prescribed or available without a prescription, as both a sleeping pill and "morning sickness" anti-nausea medication targeting pregnant women in the 1950's. The "thalidomide babies" became the case-in-point for the need of strict guidelines by the U.S. Food & Drug Administration (FDA) or full multi-species teratogenicity testing before drug approval. More recently it was found that thalidomide is useful in graft versus host disease, leprosy and resistant tuberculosis treatment, and as an anti-angiogenesis agent as a breakthrough drug for multiple myeloma (except for pregnant female patients). Decades of diabetes drug discovery research has historically focused on every possible angle, except, the energy-out side of the equation, namely, raising mitochondrial energy expenditure with chemical uncouplers. The idea of "social responsibility" allowed energy-in agents to be explored and the portfolio is robust with medicines of insulin sensitizers, insulin analogues, secretagogues, SGLT2 inhibitors, etc., but not energy-out medicines. The primary reason? It appeared unorthodox, to return to exploring a drug platform used in the 1930s in over 100,000 obese patients used for weight loss. This is over 80-years ago and prior to Dr Peter Mitchell explaining the mechanism of how mitochondrial uncouplers, like 2,4-dinitrophenol (DNP) even worked by three decades later in 1961. Although there is a clear application for metabolic disease, it was not until recently that this platform was explored for its merit at very low, weight-neutral doses, for treating insidious human illnesses and completely unrelated to weight reduction. It is known that mitochondrial uncouplers specifically target the entire organelle's physiology non-genomically. It has been known for years that many neuromuscular and neurodegenerative diseases are associated with overt production of reactive oxygen species (ROSs), a rise in isoprostanes (biomarker of mitochondrial ROSs in urine or blood) and poor calcium (Ca2+) handing. It has also been known that mitochondrial uncouplers lower ROS production and Ca2+ overload. There is evidence that elevation of isoprostanes precedes disease onset, in Alzheimer's Disease (AD). It is also curious, why so many neurodegenerative diseases of known and unknown etiology start at mid-life or later, such as Multiple Sclerosis (MS), Huntington Disease (HD), AD, Parkinson Disease, and Amyotrophic Lateral Sclerosis (ALS). Is there a relationship to a buildup of mutations that are sequestered over time due to ROSs exceeding the rate of repair? If ROS production were managed, could disease onset due to aging be delayed or prevented? Is it possible that most, if not all neurodegenerative diseases are manifested through mitochondrial dysfunction? Although DNP, a historic mitochondrial uncoupler, was used in the 1930s at high doses for obesity in well over 100,000 humans, and so far, it has never been an FDA-approved drug. This review will focus on the application of using DNP, but now, repositioned as a potential disease-modifying drug for a legion of insidious diseases at much lower and paradoxically, weight neutral doses. DNP will be addressed as a treatment for "metabesity", an emerging term related to the global comorbidities associated with the over-nutritional phenotype; obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, cardiovascular disease, but including neurodegenerative disorders and accelerated aging. Some unexpected drug findings will be discussed, such as DNP's induction of neurotrophic growth factors involved in neuronal heath, learning and cognition. For the first time in 80's years, the FDA has granted (to Mitochon Pharmaceutical, Inc., Blue Bell, PA, USA) an open Investigational New Drug (IND) approval to begin rigorous clinical testing of DNP for safety and tolerability, including for the first ever, pharmacokinetic profiling in humans. Successful completion of Phase I clinical trial will open the door to explore the merits of DNP as a possible treatment of people with many truly unmet medical needs, including those suffering from HD, MS, PD, AD, ALS, Duchenne Muscular Dystrophy (DMD), and Traumatic Brain Injury (TBI).
Asunto(s)
2,4-Dinitrofenol/metabolismo , Medicina , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Cognición , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The increased hepatic gluconeogenesis in type 2 diabetes mellitus has often been ascribed to increased transcription of phosphoenolpyruvate carboxykinase 1, cystolic form (PEPCK1), although recent evidence has questioned this attribution. To assess the metabolic role of PEPCK1, we treated regular chow fed and high-fat fed (HFF) male Sprague-Dawley rats with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) against PEPCK1 and compared them with control ASO-treated rats. PEPCK1 ASO effectively decreased PEPCK1 expression in the liver and white adipose tissue. In chow fed rats, PEPCK1 ASO did not alter adiposity, plasma glucose, or insulin. In contrast, PEPCK1 ASO decreased the white adipose tissue mass in HFF rats but without altering basal rates of lipolysis, de novo lipogenesis, or glyceroneogenesis in vivo. Despite the protection from adiposity, hepatic insulin sensitivity was impaired in HFF PEPCK1 ASO-treated rats. PEPCK1 ASO worsened hepatic steatosis, although without additional impairments in hepatic insulin signaling or activation of inflammatory signals in the liver. Instead, the development of hepatic insulin resistance and the decrease in hepatic glycogen synthesis during a hyperglycemic clamp was attributed to a decrease in hepatic glucokinase (GCK) expression and decreased synthesis of glycogen via the direct pathway. The decrease in GCK expression was associated with increased expression of activating transcription factor 3, a negative regulator of GCK transcription. These studies have demonstrated that PEPCK1 is integral to coordinating cellular metabolism in the liver and adipose tissue, although it does not directly effect hepatic glucose production or adipose glyceroneogenesis.
Asunto(s)
Adiposidad , Diabetes Mellitus Tipo 2/enzimología , Péptidos y Proteínas de Señalización Intracelular/genética , Glucógeno Hepático/biosíntesis , Hígado/metabolismo , Oligonucleótidos Antisentido/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Tejido Adiposo Blanco/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Dieta Alta en Grasa/efectos adversos , Glucoquinasa/genética , Glucoquinasa/metabolismo , Humanos , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipogénesis , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Traumatic brain injury (TBI) results in cognitive impairment, which can be long-lasting after moderate to severe TBI. Currently, there are no FDA-approved therapeutics to treat the devastating consequences of TBI and improve recovery. This study utilizes a prodrug of 2,4-dinitrophenol, MP201, a mitochondrial uncoupler with extended elimination time, that was administered after TBI to target mitochondrial dysfunction, a hallmark of TBI. Using a model of cortical impact in male C57/BL6 mice, MP201 (80 mg/kg) was provided via oral gavage 2-hr post-injury and daily afterwards. At 25-hr post-injury, mice were euthanized and the acute rescue of mitochondrial bioenergetics was assessed demonstrating a significant improvement in both the ipsilateral cortex and ipsilateral hippocampus after treatment with MP201. Additionally, oxidative markers, 4-hydroxyneneal and protein carbonyls, were reduced compared to vehicle animals after MP201 administration. At 2-weeks post-injury, mice treated with MP201 post-injury (80 mg/kg; q.d.) displayed significantly increased cortical sparing (p = .0059; 38% lesion spared) and improved cognitive outcome (p = .0133) compared to vehicle-treated mice. Additionally, vehicle-treated mice had significantly lower (p = .0019) CA3 neuron count compared to sham while MP201-treated mice were not significantly different from sham levels. These results suggest that acute mitochondrial dysfunction can be targeted to impart neuroprotection from reactive oxygen species, but chronic administration may have an added benefit in recovery. This study highlights the potential for safe, effective therapy by MP201 to alleviate negative outcomes of TBI.
Asunto(s)
2,4-Dinitrofenol/farmacología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Profármacos/farmacología , Desacopladores/farmacología , Animales , Lesiones Traumáticas del Encéfalo/inducido químicamente , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacosRESUMEN
Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of indazole-based N-alkylthiazolidenediones, which function in biochemical assays as selective inverse agonists against this receptor. Series optimization provided several potent analogues that inhibited the recruitment of a co-activator peptide fragment in vitro (IC50s < 50 nM) and reduced fasted circulating insulin and triglyceride levels in a sub-chronic pre-diabetic rat model when administered orally (10 mg/kg). A multi-parametric optimization strategy led to the identification of 50 as an advanced lead, which was more extensively evaluated in additional diabetic models. Chronic oral administration of 50 in two murine models of obesity and insulin resistance improved glucose control and reduced circulating triglycerides with efficacies similar to that of rosiglitazone. Importantly, these effects were attained without the concomitant weight gain that is typically observed with the latter agent. Thus, these studies provide additional support for the development of such molecules for the potential treatment of metabolic diseases.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/farmacología , Indazoles/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Indazoles/administración & dosificación , Indazoles/química , Ligandos , Masculino , Ratones , Ratones Obesos , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Relación Estructura-Actividad , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
The ability of novel mitochondrial uncoupler prodrug of 2,4-dinitrophenol (DNP), MP201, to prevent neuronal damage and preserve visual function in an experimental autoimmune encephalomyelitis (EAE) model of optic neuritis was evaluated. Optic nerve inflammation, demyelination, and axonal loss are prominent features of optic neuritis, an inflammatory optic neuropathy often associated with the central nervous system demyelinating disease multiple sclerosis. Currently, optic neuritis is frequently treated with high-dose corticosteroids, but treatment fails to prevent permanent neuronal damage and associated vision changes that occur as optic neuritis resolves, thus suggesting that additional therapies are required. MP201 administered orally, once per day, attenuated visual dysfunction, preserved retinal ganglion cells (RGCs), and reduced RGC axonal loss and demyelination in the optic nerves of EAE mice, with limited effects on inflammation. The prominent mild mitochondrial uncoupling properties of MP201, with slow elimination of DNP, may contribute to the neuroprotective effect by modulating the entire mitochondria's physiology directly. Results suggest that MP201 is a potential novel treatment for optic neuritis.
Asunto(s)
2,4-Dinitrofenol/metabolismo , Mitocondrias/metabolismo , Neuritis Óptica/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neuritis Óptica/metabolismo , ProfármacosRESUMEN
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the gene huntingtin. There is no treatment to prevent or delay the disease course of HD currently. Oxidative stress and mitochondrial dysfunction have emerged as key determinants of the disease progression in HD. Therefore, counteracting mutant huntingtin (mHtt)-induced oxidative stress and mitochondrial dysfunction appears as a new approach to treat this devastating disease. Interestingly, mild mitochondrial uncoupling improves neuronal resistance to stress and facilitates neuronal survival. Mild mitochondrial uncoupling can be induced by the proper dose of 2,4-dinitrophenol (DNP), a proton ionophore that was previously used for weight loss. In this study, we evaluated the effects of chronic administration of DNP at three doses (0.5, 1, 5mg/kg/day) on mHtt-induced behavioral deficits and cellular abnormalities in the N171-82Q HD mouse model. DNP at a low dose (1mg/kg/day) significantly improved motor function and preserved medium spiny neuronal marker DARPP32 and postsynaptic protein PSD95 in the striatum of HD mice. Further mechanistic study suggests that DNP at this dose reduced oxidative stress in HD mice, which was indicated by reduced levels of F2-isoprostanes in the brain of HD mice treated with DNP. Our data indicated that DNP provided behavioral benefit and neuroprotective effect at a weight neutral dose in HD mice, suggesting that the potential value of repositioning DNP to HD treatment is warranted in well-controlled clinical trials in HD.
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2,4-Dinitrofenol/farmacología , 2,4-Dinitrofenol/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/patología , Homólogo 4 de la Proteína Discs Large , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Guanilato-Quinasas/metabolismo , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Enfermedad de Huntington/fisiopatología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Actividad Motora/genética , Neuronas/metabolismo , Estrés Oxidativo/genéticaRESUMEN
Recent findings have elucidated roles for mitochondrial uncoupling proteins (UCPs) in neuronal plasticity and resistance to metabolic and oxidative stress. UCPs are induced by bioenergetic challenges such as caloric restriction and exercise and may protect neurons against dysfunction and degeneration. The pharmacological uncoupler 2,4-dinitrophenol (DNP), which was once prescribed to >100,000 people as a treatment for obesity, stimulates several adaptive cellular stress-response signaling pathways in neurons including those involving the brain-derived neurotrophic factor (BDNF), the transcription factor cyclic AMP response element-binding protein (CREB), and autophagy. Preclinical data show that low doses of DNP can protect neurons and improve functional outcome in animal models of Alzheimer's and Parkinson's diseases, epilepsy, and cerebral ischemic stroke. Repurposing of DNP and the development of novel uncoupling agents with hormetic mechanisms of action provide opportunities for new breakthrough therapeutic interventions in a range of acute and chronic insidious neurodegenerative/neuromuscular conditions, all paradoxically at body weight-preserving doses.
Asunto(s)
2,4-Dinitrofenol/administración & dosificación , Neuroprotección/fisiología , Proteína Desacopladora 1/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo , Modelos Animales de Enfermedad , Humanos , Enfermedades Neurodegenerativas/prevención & control , Plasticidad NeuronalRESUMEN
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.
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Diacilglicerol O-Acetiltransferasa/metabolismo , Pruebas de Enzimas/métodos , Glicerol/metabolismo , Hígado/enzimología , Ácido Oléico/metabolismo , Animales , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Diacilglicerol O-Acetiltransferasa/genética , Inhibidores Enzimáticos/farmacología , Esterificación/efectos de los fármacos , Ácidos Grasos/biosíntesis , Ácidos Grasos/metabolismo , Células Hep G2 , Humanos , Marcaje Isotópico , Lipoproteínas VLDL/metabolismo , Masculino , Ratones , Oligonucleótidos Antisentido/genética , Triglicéridos/biosíntesisRESUMEN
Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of diaryl ether based thiazolidenediones, which function as selective ligands against this receptor. Series optimization provided several potent analogues that inhibit the recruitment of a coactivator peptide fragment in in vitro biochemical assays (IC(50) < 150 nM) and cellular two-hybrid reporter assays against the ligand binding domain (IC(50) = 1-5 µM). A cocrystal structure of the ligand-binding domain of ERRα with lead compound 29 revealed the presence of a covalent interaction between the protein and ligand, which has been shown to be reversible. In diet-induced murine models of obesity and in an overt diabetic rat model, oral administration of 29 normalized insulin and circulating triglyceride levels, improved insulin sensitivity, and was body weight neutral. This provides the first demonstration of functional activities of an ERRα ligand in metabolic animal models.
Asunto(s)
Éteres/síntesis química , Hipoglucemiantes/síntesis química , Receptores de Estrógenos/metabolismo , Tiazolidinedionas/síntesis química , Administración Oral , Animales , Unión Competitiva , Disponibilidad Biológica , Cristalografía por Rayos X , Diabetes Mellitus/tratamiento farmacológico , Perros , Éteres/farmacocinética , Éteres/farmacología , Femenino , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Insulina/sangre , Resistencia a la Insulina , Ligandos , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Obesidad/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/genética , Relación Estructura-Actividad , Tiazolidinedionas/farmacocinética , Tiazolidinedionas/farmacología , Triglicéridos/sangre , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Statistically sound experimental design in pharmacology studies ensures that the known prognostic factors, if any, are equally represented across investigational groups to avoid bias and imbalance which could render the experiment invalid or lead to false conclusions. Complete randomization can be effective to reduce bias in the created groups especially in large sample size situations. However, in small studies which involve only few treatment subjects, as in preclinical trials, there is a high chance of imbalance. The effects of this imbalance may be removed through covariate analysis or prevented with stratified randomization, however small studies limit the number of covariates to be analyzed this way. The problem is accentuated when there are multiple baseline covariates with varying scales and magnitudes to be considered in the randomization, and creating a balanced solution becomes a combinatorial challenge. Our method, IRINI, uses an optimization technique to achieve treatment to subject group allocation across multiple prognostic factors concurrently. It ensures that the created groups are equal in size and statistically comparable in terms of mean and variance. This method is a novel application of genetic algorithms to solve the allocation problem and simultaneously ensure quality, speed of the results and randomness of the process. Results from preclinical trials demonstrate the effectiveness of the method.
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Algoritmos , Selección de Paciente , Distribución Aleatoria , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Humanos , Pronóstico , Sesgo de SelecciónRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a major contributing factor to hepatic insulin resistance in type 2 diabetes. Diacylglycerol acyltransferase (Dgat), of which there are two isoforms (Dgat1 and Dgat2), catalyzes the final step in triglyceride synthesis. We evaluated the metabolic impact of pharmacological reduction of DGAT1 and -2 expression in liver and fat using antisense oligonucleotides (ASOs) in rats with diet-induced NAFLD. Dgat1 and Dgat2 ASO treatment selectively reduced DGAT1 and DGAT2 mRNA levels in liver and fat, but only Dgat2 ASO treatment significantly reduced hepatic lipids (diacylglycerol and triglyceride but not long chain acyl CoAs) and improved hepatic insulin sensitivity. Because Dgat catalyzes triglyceride synthesis from diacylglycerol, and because we have hypothesized that diacylglycerol accumulation triggers fat-induced hepatic insulin resistance through protein kinase C epsilon activation, we next sought to understand the paradoxical reduction in diacylglycerol in Dgat2 ASO-treated rats. Within 3 days of starting Dgat2 ASO therapy in high fat-fed rats, plasma fatty acids increased, whereas hepatic lysophosphatidic acid and diacylglycerol levels were similar to those of control rats. These changes were associated with reduced expression of lipogenic genes (SREBP1c, ACC1, SCD1, and mtGPAT) and increased expression of oxidative/thermogenic genes (CPT1 and UCP2). Taken together, these data suggest that knocking down Dgat2 protects against fat-induced hepatic insulin resistance by paradoxically lowering hepatic diacylglycerol content and protein kinase C epsilon activation through decreased SREBP1c-mediated lipogenesis and increased hepatic fatty acid oxidation.
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Diacilglicerol O-Acetiltransferasa/fisiología , Hígado Graso/patología , Resistencia a la Insulina , Oligonucleótidos Antisentido/química , Animales , Diacilglicerol O-Acetiltransferasa/metabolismo , Dieta , Ácidos Grasos/metabolismo , Hígado Graso/terapia , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Oxígeno/metabolismo , Ratas , Ratas Sprague-Dawley , Triglicéridos/metabolismoRESUMEN
Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance and type 2 diabetes mellitus, but the molecular signals linking hepatic fat accumulation to hepatic insulin resistance are unknown. Three days of high-fat feeding in rats results specifically in hepatic steatosis and hepatic insulin resistance. In this setting, PKCepsilon, but not other isoforms of PKC, is activated. To determine whether PKCepsilon plays a causal role in the pathogenesis of hepatic insulin resistance, we treated rats with an antisense oligonucleotide against PKCepsilon and subjected them to 3 days of high-fat feeding. Knocking down PKCepsilon expression protects rats from fat-induced hepatic insulin resistance and reverses fat-induced defects in hepatic insulin signaling. Furthermore, we show that PKCepsilon associates with the insulin receptor in vivo and impairs insulin receptor kinase activity both in vivo and in vitro. These data support the hypothesis that PKCepsilon plays a critical role in mediating fat-induced hepatic insulin resistance and represents a novel therapeutic target for type 2 diabetes.
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Hígado Graso/enzimología , Resistencia a la Insulina , Insulina/metabolismo , Proteína Quinasa C-epsilon/fisiología , Animales , Insulina/sangre , Resistencia a la Insulina/genética , Metabolismo de los Lípidos , Masculino , Oligonucleótidos Antisentido/farmacología , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/genética , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/agonistas , Transducción de SeñalRESUMEN
Fasting hyperglycemia, a prominent finding in diabetes, is primarily due to increased gluconeogenesis. The transcription factor Foxo1 links insulin signaling to decreased transcription of PEPCK and glucose-6-phosphatase (G6Pase) and provides a possible therapeutic target in insulin-resistant states. Synthetic, optimized antisense oligonucleotides (ASOs) specifically inhibit Foxo1 expression. Here we show the effect of such therapy on insulin resistance in mice with diet-induced obesity (DIO). Reducing Foxo1 mRNA expression with ASO therapy in mouse hepatocytes decreased levels of Foxo1 protein and mRNA expression of PEPCK by 48 +/- 4% and G6Pase by 64 +/- 3%. In mice with DIO and insulin resistance, Foxo1 ASO therapy lowered plasma glucose concentration and the rate of basal endogenous glucose production. In addition, Foxo1 ASO therapy lowered both hepatic triglyceride and diacylglycerol content and improved hepatic insulin sensitivity. Foxo1 ASO also improved adipocyte insulin action. At a tissue-specific level, this manifested as improved insulin-mediated 2-deoxyglucose uptake and suppression of lipolysis. On a whole-body level, the result was improved glucose tolerance after an intraperitoneal glucose load and increased insulin-stimulated whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp. In conclusion, Foxo1 ASO therapy improved both hepatic insulin and peripheral insulin action. Foxo1 is a potential therapeutic target for improving insulin resistance.
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Factores de Transcripción Forkhead/genética , Insulina/fisiología , Hígado/fisiología , Oligonucleótidos Antisentido , Animales , Glucemia/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/deficiencia , Cinética , Ratones , Transaminasas/metabolismoRESUMEN
Hepatic steatosis is a core feature of the metabolic syndrome and type 2 diabetes and leads to hepatic insulin resistance. Malonyl-CoA, generated by acetyl-CoA carboxylases 1 and 2 (Acc1 and Acc2), is a key regulator of both mitochondrial fatty acid oxidation and fat synthesis. We used a diet-induced rat model of nonalcoholic fatty liver disease (NAFLD) and hepatic insulin resistance to explore the impact of suppressing Acc1, Acc2, or both Acc1 and Acc2 on hepatic lipid levels and insulin sensitivity. While suppression of Acc1 or Acc2 expression with antisense oligonucleotides (ASOs) increased fat oxidation in rat hepatocytes, suppression of both enzymes with a single ASO was significantly more effective in promoting fat oxidation. Suppression of Acc1 also inhibited lipogenesis whereas Acc2 reduction had no effect on lipogenesis. In rats with NAFLD, suppression of both enzymes with a single ASO was required to significantly reduce hepatic malonyl-CoA levels in vivo, lower hepatic lipids (long-chain acyl-CoAs, diacylglycerol, and triglycerides), and improve hepatic insulin sensitivity. Plasma ketones were significantly elevated compared with controls in the fed state but not in the fasting state, indicating that lowering Acc1 and -2 expression increases hepatic fat oxidation specifically in the fed state. These studies suggest that pharmacological inhibition of Acc1 and -2 may be a novel approach in the treatment of NAFLD and hepatic insulin resistance.
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Acetil-CoA Carboxilasa/antagonistas & inhibidores , Dieta , Inhibidores Enzimáticos/uso terapéutico , Hígado Graso/tratamiento farmacológico , Hígado Graso/enzimología , Resistencia a la Insulina/fisiología , Oligonucleótidos Antisentido/uso terapéutico , Acetil-CoA Carboxilasa/genética , Animales , Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Ratas , Transducción de Señal/fisiología , Triglicéridos/biosíntesisRESUMEN
Type 2 diabetes is characterized by loss of beta-cell mass and concomitant deposition of amyloid derived from islet amyloid polypeptide (IAPP). Previously we have shown that expression of human IAPP (huIAPP) in islets of transgenic mice results in either a rapid onset of hyperglycemia in mice homozygous for the huIAPP transgene on a lean background (FVB/N) or a gradual hyperglycemia in mice hemizygous for the huIAPP transgene on an obese background (A(vy)/A). In both strains, only the males routinely develop diabetes. To investigate this sexual dimorphism, we treated young prediabetic A(vy)/A mice transgenic for huIAPP (huIAPP-A(vy)) with 17beta-estradiol (E2). The treatment completely blocked the progression to hyperglycemia but also prevented the associated weight gain in these mice. Immunohistochemistry of pancreatic sections demonstrated normal islet morphology with no apparent deposition of islet amyloid. E2 treatment of 1-year-old huIAPP-A(vy) diabetic males rapidly reverses obesity and hyperglycemia. To determine the effects of E2 in a nonobese model, we also treated prediabetic, ad libitum-fed and pair-fed Lean-huIAPP transgenic males. E2 completely blocked the progression to hyperglycemia with no significant effect on body weight. Pancreatic insulin content and plasma insulin concentration of Lean-huIAPP transgenic mice increased in a dose-dependent manner. We demonstrated the presence of estrogen receptor (ER)-alpha mRNA in mouse and human islets. By also confirming the presence of ER-alpha protein in islets, we discovered a novel 58-kDa ER-alpha isoform in mice and a 52-kDa isoform in humans, in the absence of the classic 67-kDa protein found in most tissues of both species. The demonstrated presence of ER-alpha in mouse and human islets is consistent with a direct effect on islet function. We conclude that exogenous E2 administered to male mice may block human IAPP-mediated beta-cell loss both by direct action on beta-cells and by decreasing insulin demand through inhibition of weight gain or increasing insulin action.