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Homozigoto , Lipase , Proteínas de Membrana , RNA Interferente Pequeno , Humanos , Lipase/genética , RNA Interferente Pequeno/uso terapêutico , Proteínas de Membrana/genética , Hiperlipoproteinemia Tipo I/genética , Hiperlipoproteinemia Tipo I/terapia , Mutação , Masculino , Feminino , Criança , Adolescente , Adulto , Aciltransferases , Fosfolipases A2 Independentes de CálcioRESUMO
Acute Kidney Injury (AKI) is characterized by an abrupt decline in kidney function and has been associated with excess risks of death, kidney disease progression, and cardiovascular events. The kidney has a high energetic demand with mitochondrial health being essential to renal function and damaged mitochondria has been reported across AKI subtypes. 5' adenosine monophosphate-activated protein kinase (AMPK) activation preserves cellular energetics through improvement of mitochondrial function and biogenesis when ATP levels are low such as under ischemia-induced AKI. We developed a selective potent small molecule pan AMPK activator, compound 1, and tested its ability to increase AMPK activity and preserve kidney function during ischemia/reperfusion injury in rats. A single administration of 1 caused sustained activation of AMPK for at least 24 hours, protected against acute tubular necrosis, and reduced clinical markers of tubular injury such as NephroCheck and Fractional Excretion of Sodium (FENa). Reduction in plasma creatinine and increased Glomerular Filtration Rate (GFR) indicated preservation of kidney function. Surprisingly, we observed a strong diuretic effect of AMPK activation associated with natriuresis both with and without AKI. Our findings demonstrate that activation of AMPK leads to protection of tubular function under hypoxic/ischemic conditions which holds promise as a potential novel therapeutic approach for AKI. Significance Statement No approved pharmacological therapies currently exist for acute kidney injury. We developed Compound 1 which dose-dependently activated AMPK in the kidney and protected kidney function and tubules after ischemic renal injury in the rat. This was accompanied by natriuresis in injured as well as uninjured rats.
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OBJECTIVE: The mitochondrial fission protein Drp1 was proposed to promote NAFLD, as inhibition of hepatocyte Drp1 early in life prevents liver steatosis induced by high-fat diet in mice. However, whether Drp1-knockdown in older mice can reverse established NASH is unknown. METHODS: N-acetylgalactosamine-siRNA conjugates, an FDA approved method to deliver siRNA selectively to hepatocytes, were used to knockdown hepatocyte-Drp1 in mice (NAG-Drp1si). NASH was induced in C57BL/6NTac mice by Gubra-Amylin-NASH diet (D09100310, 40% fat, 22% fructose and 2% cholesterol) and treatment with NAG-Drp1si was started at week 24 of diet. Circulating transaminases, liver histology, gene expression of fibrosis and inflammation markers, and hydroxyproline synthesis determined NASH severity. Liver NEFA and triglycerides were quantified by GC/MS. Mitochondrial function was determined by respirometry. Western blots of Oma1, Opa1, p-eIf2α, as well as transcriptional analyses of Atf4-regulated genes determined ISR engagement. RESULTS: NAG-Drp1si treatment decreased body weight and induced liver inflammation in adult healthy mice. Increased hepatic Gdf15 production was the major contributor to body-weight loss caused by NAG-Drp1si treatment, as Gdf15 receptor deletion (Gfral KO) prevented the decrease in food intake and mitigated weight loss. NAG-Drp1si activated the Atf4-controlled integrated stress response (ISR) to increase hepatic Gdf15 expression. NAG-Drp1si in healthy mice caused ER stress and activated the mitochondrial protease Oma1, which are the ER and mitochondrial triggers that activate the Atf4-controlled ISR. Remarkably, induction of NASH was not sufficient to activate Oma1 in liver. However, NAG-Drp1si treatment was sufficient to activate Oma1 in adult mice with NASH, as well as exacerbating NASH-induced ER stress. Consequently, NAG-Drp1si treatment in mice with NASH led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis. CONCLUSION: Drp1 mitigates NASH by decreasing ER stress, preventing Oma1 activation and ISR exacerbation. The elevation in Gdf15 actions induced by NAG-Drp1si might represent an adaptive response decreasing the nutrient load to liver when mitochondria are misfunctional. Our study argues against blocking Drp1 in hepatocytes to combat NASH.
Assuntos
Fígado , Dinâmica Mitocondrial , Animais , Dieta Hiperlipídica/efeitos adversos , Fibrose , Inflamação/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , RNA Interferente Pequeno/metabolismo , Redução de PesoRESUMO
Peptide-based analogues of the gut-derived incretin hormone, glucagon-like peptide 1 (GLP1), stimulate insulin secretion in a glucose-dependent manner. Currently marketed GLP1 receptor (GLP1R) agonists are safe and effective in the management of Type 2 diabetes but often offer only modest weight loss. This has prompted the search for safe and effective alternatives to enhance the weight loss component of these treatments. We have demonstrated that concomitant activation GLP1R and the glucagon receptor (GCGR) can improve glucose metabolism and provide superior weight loss when compared to selective GLP1R agonism in preclinical species. This paper will highlight chemistry structure-activity relationship optimization and summarize in vivo efficacy studies toward the discovery of a once daily balanced dual agonist 12 (MK-1462), which was advanced into clinical trials.
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We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of â¼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.
Assuntos
Diabetes Mellitus Tipo 2 , Hipoglicemia , Animais , Glicemia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cães , Hipoglicemia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Receptor de Insulina , Suínos , Porco Miniatura , Índice TerapêuticoRESUMO
Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Receptor de Insulina/agonistas , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Aloxano/administração & dosagem , Aloxano/toxicidade , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Células CHO , Cricetulus , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/metabolismo , Células HEK293 , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Lipólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Ratos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Suínos , Porco MiniaturaRESUMO
Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-ß-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-ß increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-ß. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-ß induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-ß treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.
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Regulação para Baixo , Células Estreladas do Fígado/citologia , Lipase/genética , Lipase/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Idoso , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Hepatopatia Gordurosa não Alcoólica/metabolismo , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.
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Ceramidas/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Resistência à Insulina/genética , Proteínas de Membrana/genética , Oxirredutases/genética , Animais , Ceramidas/química , Ceramidas/genética , Dieta Hiperlipídica/efeitos adversos , Deleção de Genes , Leptina/deficiência , Camundongos , Camundongos Mutantes , Esfingolipídeos/química , Esfingolipídeos/metabolismoRESUMO
Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.
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Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Metabolismo Energético , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Homeostase , Camundongos Endogâmicos C57BL , Oxirredução , Condicionamento Físico AnimalRESUMO
The gut hormone PYY3-36 reduces food intake in humans and exhibits at least additive efficacy in combination with GLP-1. However, the utility of PYY analogs as anti-obesity agents has been severely limited by emesis and rapid proteolysis, a profile similarly observed with native PYY3-36 in obese rhesus macaques. Here, we found that antibody conjugation of a cyclized PYY3-36 analog achieved high NPY2R selectivity, unprecedented in vivo stability, and gradual infusion-like exposure. These properties permitted profound reduction of food intake when administered to macaques for 23 days without a single emetic event in any animal. Co-administration with the GLP-1 receptor agonist liraglutide for an additional 5 days further reduced food intake with only one animal experiencing a single bout of emesis. This antibody-conjugated PYY analog therefore may enable the long-sought potential of GLP-1/PYY-based combination treatment to achieve robust, well-tolerated weight reduction in obese patients.
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Anorexia/induzido quimicamente , Peptídeo YY/química , Peptídeo YY/farmacologia , Vômito , Animais , Células CHO , Cricetulus , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células HEK293 , Humanos , Liraglutida/farmacologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Peptídeo YY/administração & dosagem , Vômito/induzido quimicamenteRESUMO
Insulin-induced hypoglycemia in diabetes is associated with impaired glucagon secretion. In this study, we tested whether stimulation of GPR119, a G-protein-coupled receptor expressed in pancreatic islet as well as enteroendocrine cells and previously shown to stimulate insulin and incretin secretion, might enhance glucagon secretion during hypoglycemia. In the study, GPR119 agonists were applied to isolated islets or perfused pancreata to assess insulin and glucagon secretion during hypoglycemic or hyperglycemic conditions. Insulin infusion hypoglycemic clamps were performed with or without GPR119 agonist pretreatment to assess glucagon counterregulation in healthy and streptozotocin (STZ)-induced diabetic rats, including those exposed to recurrent bouts of insulin-induced hypoglycemia that leads to suppression of hypoglycemia-induced glucagon release. Hypoglycemic clamp studies were also conducted in GPR119 knockout (KO) mice to evaluate whether the pharmacological stimulatory actions of GPR119 agonists on glucagon secretion during hypoglycemia were an on-target effect. The results revealed that GPR119 agonist-treated pancreata or cultured islets had increased glucagon secretion during low glucose perfusion. In vivo, GPR119 agonists also significantly increased glucagon secretion during hypoglycemia in healthy and STZ-diabetic rats, a response that was absent in GPR119 KO mice. In addition, impaired glucagon counterregulatory responses were restored by a GPR119 agonist in STZ-diabetic rats that were exposed to antecedent bouts of hypoglycemia. Thus, GPR119 agonists have the ability to pharmacologically augment glucagon secretion, specifically in response to hypoglycemia in diabetic rodents. Whether this effect might serve to diminish the occurrence and severity of iatrogenic hypoglycemia during intensive insulin therapy in patients with diabetes remains to be established.
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Glucagon/metabolismo , Hipoglicemia/induzido quimicamente , Hipoglicemia/metabolismo , Insulina/efeitos adversos , Receptores Acoplados a Proteínas G/agonistas , Adulto , Animais , Células Cultivadas , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Estreptozocina , Adulto JovemRESUMO
Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.
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Glucose/metabolismo , Hipoglicemia/tratamento farmacológico , Insulina/farmacologia , Animais , Antígenos CD , Glicemia , Linhagem Celular , Diabetes Mellitus Tipo 2 , Modelos Animais de Doenças , Hipoglicemiantes/farmacologia , Lectinas Tipo C/efeitos dos fármacos , Fígado/patologia , Macrófagos , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Receptor de Insulina/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacosRESUMO
An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.
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Óxido de Deutério/farmacologia , Ácido Palmítico/sangue , Acetatos/sangue , Adipogenia , Animais , Feminino , Meia-Vida , Lipogênese/efeitos dos fármacos , Macaca mulatta , Masculino , Camundongos Endogâmicos C57BLRESUMO
Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by â¼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).
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Diabetes Mellitus Tipo 1/tratamento farmacológico , Desenho de Fármacos , Hipoglicemiantes/uso terapêutico , Insulina Regular Humana/análogos & derivados , Lectinas Tipo C/agonistas , Lectinas de Ligação a Manose/agonistas , Receptor de Insulina/agonistas , Receptores de Superfície Celular/agonistas , Animais , Animais Endogâmicos , Ligação Competitiva , Células CHO , Cricetulus , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Hiperglicemia/prevenção & controle , Hipoglicemia/induzido quimicamente , Hipoglicemia/prevenção & controle , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacocinética , Insulina Regular Humana/efeitos adversos , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/uso terapêutico , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligantes , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Taxa de Depuração Metabólica , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Suínos , Porco MiniaturaRESUMO
Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.
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Descoberta de Drogas/métodos , Análise do Fluxo Metabólico/métodos , Animais , Humanos , Marcação por Isótopo , Isótopos/química , Água/química , Água/metabolismoRESUMO
5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.
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Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomegalia/induzido quimicamente , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Imidazóis/farmacologia , Piridinas/farmacologia , Animais , Benzimidazóis , Glicemia/efeitos dos fármacos , Jejum , Glicogênio/metabolismo , Hipoglicemia/induzido quimicamente , Imidazóis/efeitos adversos , Imidazóis/química , Insulina/farmacologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Piridinas/efeitos adversos , Piridinas/químicaRESUMO
GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.
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Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Lipólise , Receptores Acoplados a Proteínas G/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Resistência à Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiopatologia , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Ratos , Receptores Acoplados a Proteínas G/agonistasRESUMO
Insulin resistance and diabetes can develop spontaneously with obesity and aging in rhesus monkeys, highly similar to the natural history of obesity, insulin resistance, and progression to type 2 diabetes in humans. The current studies in obese rhesus were undertaken to assess hepatic and adipose contributions to systemic insulin resistance-currently, a gap in our knowledge-and to benchmark the responses to pioglitazone (PIO). A two-step hyperinsulinemic-euglycemic clamp, with tracer-based glucose flux estimates, was used to measure insulin resistance, and in an intervention study was repeated following 6 wk of PIO treatment (3 mg/kg). Compared with lean healthy rhesus, obese rhesus has a 60% reduction of glucose utilization during a high insulin infusion and markedly impaired suppression of lipolysis, which was evident at both low and high insulin infusion. However, obese dysmetabolic rhesus manifests only mild hepatic insulin resistance. Six-week PIO treatment significantly improved skeletal muscle and adipose insulin resistance (by ~50%). These studies strengthen the concept that insulin resistance in obese rhesus closely resembles human insulin resistance and indicate the value of obese rhesus for appraising new insulin-sensitizing therapeutics.