RESUMEN
Microphysiological systems (MPS) are powerful tools for emulating human physiology and replicating disease progression in vitro. MPS could be better predictors of human outcome than current animal models, but mechanistic interpretation and in vivo extrapolation of the experimental results remain significant challenges. Here, we address these challenges using an integrated experimental-computational approach. This approach allows for in silico representation and predictions of glucose metabolism in a previously reported MPS with two organ compartments (liver and pancreas) connected in a closed loop with circulating medium. We developed a computational model describing glucose metabolism over 15 days of culture in the MPS. The model was calibrated on an experiment-specific basis using data from seven experiments, where HepaRG single-liver or liver-islet cultures were exposed to both normal and hyperglycemic conditions resembling high blood glucose levels in diabetes. The calibrated models reproduced the fast (i.e. hourly) variations in glucose and insulin observed in the MPS experiments, as well as the long-term (i.e. over weeks) decline in both glucose tolerance and insulin secretion. We also investigated the behaviour of the system under hypoglycemia by simulating this condition in silico, and the model could correctly predict the glucose and insulin responses measured in new MPS experiments. Last, we used the computational model to translate the experimental results to humans, showing good agreement with published data of the glucose response to a meal in healthy subjects. The integrated experimental-computational framework opens new avenues for future investigations toward disease mechanisms and the development of new therapies for metabolic disorders.
Asunto(s)
Diabetes Mellitus , Insulina , Animales , Humanos , Insulina/metabolismo , Glucosa/metabolismo , Diabetes Mellitus/metabolismo , Hígado/metabolismo , Secreción de Insulina , Glucemia/metabolismoRESUMEN
A synthetic protocol for 34 S-labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS-based assay analysis. This was enabled by a highly efficient, two-step, one-pot synthesis of 34 S-labeled phenylacetyl disulfide (34 S-PADS), starting from 34 S-enriched elemental sulfur (34 S8 ). 34 S-PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone. The 34 S-SIL PS ONs are shown to retain the same melting temperature, antisense activity, and secondary structure as those of the corresponding unlabeled 32 S PS ONs.
Asunto(s)
Oligonucleótidos Antisentido , Fenilacetatos , Oligonucleótidos Fosforotioatos , Sulfuros , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células HEK293 , Humanos , Marcaje Isotópico , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/química , Fenilacetatos/síntesis química , Fenilacetatos/química , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/química , ARN Largo no Codificante/metabolismo , Sulfuros/síntesis química , Sulfuros/química , Azufre/químicaRESUMEN
Current research on metabolic disorders and diabetes relies on animal models because multi-organ diseases cannot be well studied with standard in vitro assays. Here, we have connected cell models of key metabolic organs, the pancreas and liver, on a microfluidic chip to enable diabetes research in a human-based in vitro system. Aided by mechanistic mathematical modeling, we demonstrate that hyperglycemia and high cortisone concentration induce glucose dysregulation in the pancreas-liver microphysiological system (MPS), mimicking a diabetic phenotype seen in patients with glucocorticoid-induced diabetes. In this diseased condition, the pancreas-liver MPS displays beta-cell dysfunction, steatosis, elevated ketone-body secretion, increased glycogen storage, and upregulated gluconeogenic gene expression. Conversely, a physiological culture condition maintains glucose tolerance and beta-cell function. This method was reproducible in two laboratories and was effective in multiple pancreatic islet donors. The model also provides a platform to identify new therapeutic proteins, as demonstrated with a combined transcriptome and proteome analysis.
Asunto(s)
Cortisona , Glucosa , Homeostasis , Hígado , Páncreas , Humanos , Hígado/metabolismo , Hígado/efectos de los fármacos , Cortisona/metabolismo , Glucosa/metabolismo , Páncreas/metabolismo , Dispositivos Laboratorio en un Chip , Células Secretoras de Insulina/metabolismo , Sistemas MicrofisiológicosRESUMEN
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
RESUMEN
AIMS/HYPOTHESIS: GPR44 (DP2, PTGDR2, CRTh2) is the receptor for the pro-inflammatory mediator prostaglandin D2 (PGD2) and it is enriched in human islets. In rodent islets, PGD2 is produced in response to glucose, suggesting that the PGD2-GPR44/DP2 axis may play a role in human islet function during hyperglycemia. Consequently, the aim of this work was to elucidate the insulinotropic role of GPR44 antagonism in vitro in human beta-cells and in type 2 diabetes (T2DM) patients. METHODS: We determined the drive on PGD2 secretion by glucose and IL-1beta, as well as, the impact on insulin secretion by pharmacological GPR44/DP2 antagonism (AZD1981) in human islets and beta-cells in vitro. To test if metabolic control would be improved by antagonizing a hyperglycemia-driven increased PGD2 tone, we performed a proof-of-mechanism study in 20 T2DM patients (average 54 years, HbA1c 9.4%, BMI 31.6 kg/m2). The randomized, double-blind, placebo-controlled cross-over study consisted of two three-day treatment periods (AZD1981 or placebo) separated by a three-day wash-out period. Mixed meal tolerance test (MMTT) and intravenous graded glucose infusion (GGI) was performed at start and end of each treatment period. Assessment of AZD1981 pharmacokinetics, glucose, insulin, C-peptide, glucagon, GLP-1, and PGD2 pathway biomarkers were performed. RESULTS: We found (1) that PGD2 is produced in human islet in response to high glucose or IL-1beta, but likely by stellate cells rather than endocrine cells; (2) that PGD2 suppresses both glucose and GLP-1 induced insulin secretion in vitro; and (3) that the GPR44/DP2 antagonist (AZD1981) in human beta-cells normalizes insulin secretion. However, AZD1981 had no impact on neither glucose nor incretin dependent insulin secretion in humans (GGI AUC C-peptide 1-2h and MMTT AUC Glucose 0-4h LS mean ratios vs placebo of 0.94 (80% CI of 0.90-0.98, p = 0.12) and 0.99 (90% CI of 0.94-1.05, p = 0.45), despite reaching the expected antagonist exposure. CONCLUSION/INTERPRETATION: Pharmacological inhibition of the PGD2-GPR44/DP2 axis has no major impact on the modulation of acute insulin secretion in T2DM patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02367066.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Prostaglandina D2/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Factores de Transcripción/metabolismo , Acetatos/farmacología , Acetatos/uso terapéutico , Glucemia/metabolismo , Péptido C/sangre , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Persona de Mediana Edad , Prostaglandina D2/antagonistas & inhibidores , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Human in vitro physiological models studying disease and drug treatment effects are urgently needed as more relevant tools to identify new drug targets and therapies. We have developed a human microfluidic two-organ-chip model to study pancreatic islet-liver cross-talk based on insulin and glucose regulation. We have established a robust co-culture of human pancreatic islet microtissues and liver spheroids maintaining functional responses up to 15 days in an insulin-free medium. Functional coupling, demonstrated by insulin released from the islet microtissues in response to a glucose load applied in glucose tolerance tests on different days, promoted glucose uptake by the liver spheroids. Co-cultures maintained postprandial glucose concentrations in the circulation whereas glucose levels remained elevated in both single cultures. Thus, insulin secreted into the circulation stimulated glucose uptake by the liver spheroids, while the latter, in the absence of insulin, did not consume glucose as efficiently. As the glucose concentration fell, insulin secretion subsided, demonstrating a functional feedback loop between the liver and the insulin-secreting islet microtissues. Finally, inter-laboratory validation verified robustness and reproducibility. Further development of this model using tools inducing impaired glucose regulation should provide a unique in vitro system emulating human type 2 diabetes mellitus.
Asunto(s)
Técnicas de Cocultivo , Diabetes Mellitus Tipo 2/fisiopatología , Islotes Pancreáticos/fisiopatología , Hígado/fisiopatología , Esferoides Celulares/fisiología , Técnicas de Cultivo de Tejidos , Línea Celular , Técnicas de Cocultivo/instrumentación , Medios de Cultivo/análisis , Diabetes Mellitus Tipo 2/patología , Diseño de Equipo , Retroalimentación Fisiológica , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Islotes Pancreáticos/patología , Hígado/patología , Modelos Biológicos , Reproducibilidad de los Resultados , Esferoides Celulares/patología , Técnicas de Cultivo de Tejidos/instrumentaciónRESUMEN
In a world with increasing incidences of obesity, it becomes critical to understand the detailed regulation of appetite. To identify novel regulators of the signaling mediated by one of the key hormones of energy homeostasis, leptin, we screened a set of compounds for their effect on the downstream Signal Transducer and Activator of Transcription 3 (STAT3) signaling. Interestingly, cells exposed to inhibitors of the Ataxia Telangiectasia and RAD3-related protein ATR increased their leptin dependent STAT3 activity. This was due to failure of the cells to induce the negative feedback mediator Suppressor of Cytokine Signaling 3 (SOCS3), suggesting that ATR has a previously unknown role in the negative feedback regulation of leptin signaling. This is an important finding not only because it sheds light on additional genes involved in leptin signaling, but also because it brings forward a new potential therapeutic intervention point for increasing leptin signaling in obese individuals.
Asunto(s)
Leptina/fisiología , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Retroalimentación Fisiológica , Expresión Génica , Células HEK293 , Humanos , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired ß-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic ß-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve ß-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.