RESUMO
Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.
Assuntos
Glucagon/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Tri-Iodotironina/efeitos dos fármacos , Animais , Aterosclerose/tratamento farmacológico , Peso Corporal/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Engenharia Química/métodos , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Glucagon/efeitos adversos , Glucagon/química , Glucagon/farmacologia , Hiperglicemia/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Terapia de Alvo Molecular , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Tri-Iodotironina/efeitos adversos , Tri-Iodotironina/química , Tri-Iodotironina/farmacologiaRESUMO
BACKGROUND: Pharmacotherapies targeting motivational aspects of feeding and palatable food reward, while sparing mood and cognitive function, represent an alluring approach to reverse obesity and maintain weight loss in an obesogenic environment. A novel glucagon-like peptide-1/dexamethasone (GLP-1/Dexa) conjugate, developed to selectively activate glucocorticoid receptors in GLP-1 receptor-expressing cells was shown to decrease food intake and lower body weight in obese mice. Here, we investigate if this novel drug candidate modulates the rewarding properties of food and if it affects behavioral indices of mood and memory. METHODS: C57Bl6 mice treated with the GLP-1/Dexa conjugate, GLP-1 or vehicle lever-pressed for high-fat, high sugar (HFHS) food rewards in an operant task. Alterations in food-motivated behavior were also assessed following a HFHS diet withdrawal manipulation (switch to chow). The effects of repeated GLP-1/Dexa conjugate, GLP-1 or vehicle on free-feeding intake, body weight, anxiodepressive behaviors (elevated-plus maze, open field test & forced swim test), memory (novel object recognition) and mRNA expression of reward-relevant markers in the nucleus accumbens were also evaluated in mice fed a HFHS diet for 12 weeks. RESULTS: Mice treated with a GLP-1 analogue displayed a transient (4â¯h) reduction in their motivation to lever press for HFHS reward, whereas treatment with equimolar doses of GLP-1/Dexa delivered a superior and sustained (20â¯h) suppression of food-motivated behavior. GLP-1/Dexa also inhibited food reward following withdrawal from the HFHS diet. These benefits coincided with related transcriptional changes of dopaminergic markers in the nucleus accumbens. Importantly, repeated GLP-1/Dexa treatment during a HFHS diet caused weight loss without affecting anxiodepressive behavior and memory. CONCLUSION: Via its actions to blunt the rewarding effects of palatable food without affecting mood and recognition memory, GLP-1-directed targeting of dexamethasone may serve as a promising and safe anti-obesity strategy.
Assuntos
Afeto/efeitos dos fármacos , Dexametasona/farmacologia , Alimentos , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Memória/efeitos dos fármacos , Motivação/efeitos dos fármacos , Recompensa , Animais , Condicionamento Operante/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , CamundongosRESUMO
OBJECTIVE: Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. METHODS: We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. RESULTS: Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. CONCLUSIONS: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.
Assuntos
AMP Cíclico/biossíntese , Endossomos/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Endocitose/fisiologia , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Lisossomos/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Sistemas do Segundo Mensageiro , Transdução de SinaisRESUMO
Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity.
Assuntos
Dexametasona/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Glucocorticoides/uso terapêutico , Incretinas/uso terapêutico , Inflamação/tratamento farmacológico , Obesidade/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Dexametasona/análogos & derivados , Metabolismo Energético/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Glucocorticoides/química , Glucose/metabolismo , Células HEK293 , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Incretinas/química , Inflamação/complicações , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicações , Obesidade/metabolismoRESUMO
A rapid and specific liquid chromatographic mass spectrometric (LC-MS-MS) method has been developed for the determination of paroxetine in human plasma. The procedure involves a liquid-liquid extraction of paroxetine and fluoxetine (internal standard) with cyclohexane-ethyl acetate. The standard curve was linear over a working range of 0.2-50 ng/ml. The lower limit of quantitation was 0.2 ng/ml. No endogenous compounds were found to interfere with the analysis. The absolute recovery was 70.8% for paroxetine and 84.1% for the internal standard. The accuracy of inter-assay and intra-assay accuracy was in the ranges -4.8 to -0.5% and -3.4 to 4.8%, respectively. This method proved to be suitable for bioequivalence studies by being simple, selective and reproducible.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Paroxetina/sangue , Inibidores Seletivos de Recaptação de Serotonina/sangue , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A new method, using high-performance liquid chromatography/ion electrospray (negative ion) mass spectrometry, has been developed for the determination of a hydrophilic liver-specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin in human plasma. In this method, plasma samples were prepared by a solid-phase extraction on C(18) Bond Elut cartridge. Chromatography was carried out with a Zorbax C(8) column. Simple isocratic chromatography conditions were used. The method has been validated in a linear range of 0.25-300 ng/ml with a coefficient of variation of 0.6-3.4%. The overall recovery was 90.5% for pravastatin and 90.8% for the internal standard beta-hydroxy-lovastatin. The method is simple and reliable with a total run time of less than 2 min.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/sangue , Espectrometria de Massas/métodos , Pravastatina/sangue , Humanos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
A fast and robust liquid chromatography-mass spectrometry (LC-MS-MS) method has been developed for simultaneous quantitation of the angiotensin-converting enzyme (ACE) inhibitor, ramipril and its metabolite ramiprilat in human plasma. The method involves a solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables a detection limit at sub-nanogram levels. The proposed method has been validated with a linear range of 0.5-250 ng/ml for both ramipril and ramiprilat. The overall recoveries for ramipril and ramiprilat were 88.7 and 101.8%, respectively.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Ramipril/sangue , Inibidores da Enzima Conversora de Angiotensina/farmacocinética , Humanos , Ramipril/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A simple and reproducible high performance liquid chromatography-tandem mass spectrometric method was developed for methocarbamol analysis in human plasma. Methocarbamol and the internal standard (IS) were extracted by a protein precipitation method. Under isocratic separation condition the chromatographic run time was 3.0 min. The calibration curve was linear over a range of 150-12,000 ng/mL with good intraday assay and interday assay precision (CV%<10.9%). The method was proven to be sensitive and selective for the analysis of methocarbamol in human plasma for bioequivalence study.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metocarbamol/sangue , Relaxantes Musculares Centrais/sangue , Espectrometria de Massas em Tandem/métodos , HumanosRESUMO
This event was organized by the Calibration and Validation Group (a scientific nonprofit organization based in Toronto, Canada) as a 1.5-day workshop for contract research organizations and pharmaceutical companies involved in providing bioanalytical data for bioavailability, bioequivalence, pharmacokinetic and comparability studies.