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Glucagon-like peptide 1 (GLP-1) is secreted from enteroendocrine L-cells in response to oral nutrient intake and elicits glucose-stimulated insulin secretion while suppressing glucagon secretion. It also slows gastric emptying, which contributes to decreased postprandial glycemic excursions. In the 1990s, chronic subcutaneous infusion of GLP-1 was found to lower blood glucose levels in patients with type 2 diabetes. However, GLP-1's very short half-life, arising from cleavage by the enzyme dipeptidyl peptidase 4 (DPP-4) and glomerular filtration by the kidneys, presented challenges for clinical use. Hence, DPP-4 inhibitors were developed, as well as several GLP-1 analogs engineered to circumvent DPP-4-mediated breakdown and/or rapid renal elimination. Three categories of GLP-1 analogs, are being developed and/or are in clinical use: short-acting, long-acting, and prolonged-acting GLP-1 analogs. Each class has different plasma half-lives, molecular size, and homology to native GLP-1, and consequently different characteristic effects on glucose metabolism. In this article, we review current clinical data derived from each class of GLP-1 analogs, and consider the clinical effects reported for each category in recent head to head comparison studies. Given the relatively brief clinical history of these compounds, we also highlight several important efficacy and safety issues which will require further investigation.
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OBJECTIVE: Leptin therapy has been found to reverse hyperglycemia and prevent mortality in several rodent models of type 1 diabetes. Yet the mechanism of leptin-mediated reversal of hyperglycemia has not been fully defined. The liver is a key organ regulating glucose metabolism and is also a target of leptin action. Thus we hypothesized that exogenous leptin administered to mice with streptozotocin (STZ)-induced diabetes reverses hyperglycemia through direct action on hepatocytes. RESEARCH DESIGN AND METHODS: After the induction of diabetes in mice with a high dose of STZ, recombinant mouse leptin was delivered at a supraphysiological dose for 14 days by an osmotic pump implant. We characterized the effect of leptin administration in C57Bl/6J mice with STZ-induced diabetes and then examined whether leptin therapy could reverse STZ-induced hyperglycemia in mice in which hepatic leptin signaling was specifically disrupted. RESULTS: Hyperleptinemia reversed hyperglycemia and hyperketonemia in diabetic C57Bl/6J mice and dramatically improved glucose tolerance. These effects were associated with reduced plasma glucagon and growth hormone levels and dramatically enhanced insulin sensitivity, without changes in glucose uptake by skeletal muscle. Leptin therapy also ameliorated STZ-induced hyperglycemia and hyperketonemia in mice with disrupted hepatic leptin signaling to a similar extent as observed in wild-type littermates with STZ-induced diabetes. CONCLUSIONS: These observations reveal that hyperleptinemia reverses the symptoms of STZ-induced diabetes in mice and that this action does not require direct leptin signaling in the liver.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Leptina/uso terapéutico , Hígado/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Glucemia/efectos de los fármacos , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/metabolismo , Ensayo de Inmunoadsorción Enzimática , Glucagón/sangre , Hormona del Crecimiento/sangre , Hiperglucemia/sangre , Leptina/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Periodo Posprandial , Receptores de Leptina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS: Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS: A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other ß-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS: These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to ß-cells.
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Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Células Secretoras de Glucagón/citología , Glucagón/metabolismo , Animales , Glucemia/metabolismo , Línea Celular , ADN/análisis , ADN/genética , Células Madre Embrionarias/metabolismo , Glucagón/análisis , Células Secretoras de Glucagón/metabolismo , Humanos , Inmunohistoquímica , Insulina/análisis , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Proglucagón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Islet transplantation represents a potential cure for type 1 diabetes; however, a lack of sufficient donor material limits its clinical use. To address the shortfall of islet availability, surrogate insulin-producing cells are sought. Studies suggest that human amniotic fluid (hAF) contains multipotent progenitor cells capable of differentiating to all three germ layers. Here, we used high-content, live-cell imaging to assess the ability to reprogram hAF cells towards a beta cell phenotype. A fluorescent reporter system was developed where DsRed express (DSRE) expression is driven by the human insulin promoter. Using integrative lentiviral technology, we created stable reporter hAF cells that could be routinely monitored for insulin promoter activation. These cells were subjected to combinatorial high-content screening using adenoviral-mediated expression of up to six transcription factors important for beta cell development. Cells were monitored for DSRE expression which revealed an optimal combination of the transcription factors required to induce insulin gene expression in hAF cells. These optimally induced cells were examined for expression of additional beta cell transcription factors and proteins involved in glucose sensing and insulin processing. RT-qPCR revealed very low level expression of insulin that was ultimately insufficient to reverse streptozotocin-induced diabetes following sub-capsular kidney transplantation. High-content, live-cell imaging using fluorescent reporter cells provides a convenient method for repeated assessment of cellular reprogramming. hAF cells could be reprogrammed to express key beta cell proteins, however insulin gene expression was insufficient to reverse hyperglycemia in diabetic animals.
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Líquido Amniótico/citología , Insulina/metabolismo , Adenoviridae/genética , Animales , Células Cultivadas , Citometría de Flujo , Genes Reporteros , Humanos , Inmunohistoquímica , Insulina/genética , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Regiones Promotoras Genéticas , Ratas , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND & AIMS: Glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon product glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion. Interestingly, a subset of enteroendocrine cells express both GIP and GLP-1. We sought to determine whether GIP also might be co-expressed with proglucagon in pancreatic alpha-cells. METHODS: We assessed GIP expression via reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. We developed a novel bioassay to measure GIP release from isolated islets, compared the biological activities of full-length and truncated GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. RESULTS: GIP messenger RNA was present in mouse islets; GIP protein localized to islet alpha-cells of mouse, human, and snake pancreas, based on immunohistochemical analyses. However, using a C-terminal GIP antibody, immunoreactivity was detected in islets from prohormone convertase (PC) 2 knockout but not wild-type mice. Bioactive GIP was secreted from mouse and human islets after arginine stimulation. In the perfused mouse pancreas, GIP(1-42) and amidated GIP(1-30) had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. CONCLUSIONS: GIP is expressed in and secreted from pancreatic islets; in alpha-cells, PC2 processes proGIP to yield a truncated but bioactive form of GIP that differs from the PC1/3-derived form from K-cells. Islet-derived GIP promotes islet glucose competence and also could support islet development and/or survival.
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Polipéptido Inhibidor Gástrico/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Boidae , Línea Celular , Duodeno/metabolismo , Femenino , Polipéptido Inhibidor Gástrico/genética , Péptido 1 Similar al Glucagón/metabolismo , Humanos , Secreción de Insulina , Islotes Pancreáticos/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Proglucagón/metabolismo , Proproteína Convertasa 2/deficiencia , Proproteína Convertasa 2/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , TransfecciónRESUMEN
The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are produced predominantly by enteroendocrine cells and have multiple blood glucose-lowering effects. Recent years have seen a surge of interest in understanding the basic physiology and pathophysiology of incretins and in applying this knowledge to the treatment of diabetes and obesity. Considerable gains have been made in elucidating the mechanisms controlling incretin secretion, and there is growing evidence to suggest that incretins might be involved in the rapid reversal of diabetes observed in gastric bypass patients. Here, we review these recent advances and outline the multiple strategies being pursued to exploit the potential therapeutic benefits of GIP and GLP-1.
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Incretinas/fisiología , Animales , Cirugía Bariátrica , Diabetes Mellitus/terapia , Inhibidores de la Dipeptidil-Peptidasa IV , Descubrimiento de Drogas , Ingestión de Alimentos/fisiología , Células Enteroendocrinas/metabolismo , Polipéptido Inhibidor Gástrico/biosíntesis , Polipéptido Inhibidor Gástrico/metabolismo , Polipéptido Inhibidor Gástrico/fisiología , Péptido 1 Similar al Glucagón/biosíntesis , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/fisiología , Humanos , Hipoglucemiantes , Incretinas/biosíntesis , Incretinas/metabolismo , Islotes Pancreáticos/metabolismo , Imitación Molecular , Obesidad/terapiaRESUMEN
Most patients with type 1 diabetes rely on multiple daily insulin injections to maintain blood glucose control. However, insulin injections carry the risk of inducing hypoglycemia and do not eliminate diabetic complications. We sought to develop and evaluate a regulatable cell-based system for delivery of insulin to treat diabetes. We generated two intestinal cell lines in which human insulin expression is controlled by mifepristone. Insulin mRNA expression was dependent on the mifepristone dose and incubation time and cells displayed insulin and C-peptide immunoreactivity and glucose-induced insulin release following mifepristone treatment. Cell transplantation followed by mifepristone administration reversed streptozotocin (STZ)-induced diabetes in mice, and this effect was dependent on the mifepristone dose delivered. These data support the notion that engineering regulatable insulin expression within a cell already equipped for regulated secretion may be efficacious for the treatment of insulin-dependent diabetes.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Diabetes Mellitus Experimental/terapia , Regulación de la Expresión Génica/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Mifepristona/farmacología , Animales , Línea Celular Tumoral/trasplante , Humanos , Intestinos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are released during meals from endocrine cells located in the gut mucosa and stimulate insulin secretion from pancreatic beta-cells in a glucose-dependent manner. Although the gut epithelium senses luminal sugars, the mechanism of sugar sensing and its downstream events coupled to the release of the incretin hormones are not clearly elucidated. Recently, it was reported that sucralose, a sweetener that activates the sweet receptors of taste buds, triggers incretin release from a murine enteroendocrine cell line in vitro. We confirmed that immunoreactivity of alpha-gustducin, a key G-coupled protein involved in taste sensing, is sometimes colocalized with GIP in rat duodenum. We investigated whether secretion of incretins in response to carbohydrates is mediated via taste receptors by feeding rats the sweet-tasting compounds saccharin, acesulfame potassium, d-tryptophan, sucralose, or stevia. Oral gavage of these sweeteners did not reduce the blood glucose excursion to a subsequent intraperitoneal glucose tolerance test. Neither oral sucralose nor oral stevia reduced blood glucose levels in Zucker diabetic fatty rats. Finally, whereas oral glucose increased plasma GIP levels approximately 4-fold and GLP-1 levels approximately 2.5-fold postadministration, none of the sweeteners tested significantly increased levels of these incretins. Collectively, our findings do not support the concept that release of incretins from enteroendocrine cells is triggered by carbohydrates via a pathway identical to the sensation of "sweet taste" in the tongue.
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Sacarosa en la Dieta/farmacología , Duodeno/metabolismo , Péptido 1 Similar al Glucagón/sangre , Incretinas/sangre , Edulcorantes/farmacología , Administración Oral , Animales , Polipéptido Inhibidor Gástrico/sangre , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Wistar , Ratas Zucker , Sacarina/farmacología , Stevia , Sacarosa/análogos & derivados , Sacarosa/farmacología , Tiazinas/farmacología , Transducina/metabolismo , Triptófano/farmacologíaRESUMEN
Type 2 diabetes (T2D) is characterized by elevated blood glucose levels owing to insufficient secretion and/or activity of the glucose-lowering hormone insulin. Glucagon-like peptide-1 (GLP-1) has received much attention as a new treatment for diabetes because of its multiple blood glucose-lowering effects, including glucose-dependent enhancement of insulin secretion, inhibition of gastric emptying, and promotion of the survival and growth of insulin-producing beta-cells. GLP-1, along with GLP-2 and oxyntomodulin, is produced in the intestinal L-cell via processing of proglucagon by prohormone convertase 1/3 (PC1/3), while in the pancreatic alpha-cell, coexpression of proglucagon and the alternate enzyme PC2 typically results in differential processing of proglucagon to yield glucagon. We used alginate-encapsulated alpha-cells as a model to evaluate continuous delivery of PC1/3- or PC2-derived proglucagon products. In high fat-fed and db/db mice, PC1/3-, but not PC2-expressing alpha-cells improved glucose handling and transiently lowered fasting glucose levels, suggesting that continuous delivery of PC1/3-derived proglucagon products via cell therapy may be useful for diabetes treatment. In addition, we show that long-term treatment with PC1/3-expressing, but not PC2-expressing, alpha-cells improved cold-induced thermogenesis in db/db mice, demonstrating a previously unappreciated effect of one or more PC1/3-derived alpha-cell products.
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Frío , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Glucagón/trasplante , Glucosa/metabolismo , Proproteína Convertasa 1/metabolismo , Animales , Composición Corporal , Células Cultivadas , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/terapia , Glucagón/metabolismo , Leptina/farmacología , Ratones , Proglucagón/metabolismo , Proproteína Convertasa 2/metabolismoRESUMEN
OBJECTIVE: Glucagon, which raises blood glucose levels by stimulating hepatic glucose production, is produced in alpha-cells via cleavage of proglucagon by prohormone convertase (PC)-2. In the enteroendocrine L-cell, proglucagon is differentially processed by the alternate enzyme PC1/3 to yield glucagon-like peptide (GLP)-1, GLP-2, and oxyntomodulin, which have blood glucose-lowering effects. We hypothesized that alteration of PC expression in alpha-cells might convert the alpha-cell from a hyperglycemia-promoting cell to one that would improve glucose homeostasis. RESEARCH DESIGN AND METHODS: We compared the effect of transplanting encapsulated PC2-expressing alpha TC-1 cells with PC1/3-expressing alpha TCDeltaPC2 cells in normal mice and low-dose streptozotocin (STZ)-treated mice. RESULTS: Transplantation of PC2-expressing alpha-cells increased plasma glucagon levels and caused mild fasting hyperglycemia, impaired glucose tolerance, and alpha-cell hypoplasia. In contrast, PC1/3-expressing alpha-cells increased plasma GLP-1/GLP-2 levels, improved glucose tolerance, and promoted beta-cell proliferation. In GLP-1R(-/-) mice, the ability of PC1/3-expressing alpha-cells to improve glucose tolerance was attenuated. Transplantation of PC1/3-expressing alpha-cells prevented STZ-induced hyperglycemia by preserving beta-cell area and islet morphology, possibly via stimulating beta-cell replication. However, PC2-expressing alpha-cells neither prevented STZ-induced hyperglycemia nor increased beta-cell proliferation. Transplantation of alpha TCDeltaPC2, but not alpha TC-1 cells, also increased intestinal epithelial proliferation. CONCLUSIONS: Expression of PC1/3 rather than PC2 in alpha-cells induces GLP-1 and GLP-2 production and converts the alpha-cell from a hyperglycemia-promoting cell to one that lowers blood glucose levels and promotes islet survival. This suggests that alteration of proglucagon processing in the alpha-cell may be therapeutically useful in the context of diabetes.
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Células Secretoras de Glucagón/enzimología , Células Secretoras de Glucagón/trasplante , Glucosa/metabolismo , Proglucagón/metabolismo , Proproteína Convertasa 1/genética , Proproteína Convertasa 2/genética , Animales , Supervivencia Celular , Diabetes Mellitus Experimental/terapia , Receptor del Péptido 1 Similar al Glucagón , Células Secretoras de Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Noqueados , Proproteína Convertasa 2/deficiencia , Receptores de Glucagón/deficienciaRESUMEN
PURPOSE OF REVIEW: Islet transplantation is being used to treat type 1 diabetes but is currently limited by the shortage of tissue available and by insufficient long-term function of transplanted islets. Thus, there remains significant interest in developing substitute sources of insulin-producing cells. Here we review progress in this area, focusing on insulin gene therapy and generation of new insulin-producing cells by redirecting hepatic and intestinal tissues towards a ß-cell phenotype. RECENT FINDINGS: Insulin gene therapy using non-ß cells has been improved by utilizing modified insulin constructs controlled by regulatory elements to confer nutrient responsiveness, and by inducing insulin production in endocrine cells that are equipped for rapid and in some cases glucose-responsive secretion. Significant advances have also been made towards generation of insulin-producing cells via transcriptional manipulation of hepatic and intestinal cells. These approaches offer the potential of generating a virtually limitless supply of insulin-producing cells. SUMMARY: The major challenge associated with insulin gene therapy in non-ß cells is to achieve rapid, glucose-responsive secretion, while transdifferentiation approaches require additional characterization of the function and stability of insulin-producing cells. Continued efforts in these areas are warranted, as re-establishment of endogenous insulin production would be a welcome replacement to insulin injections for diabetes treatment.
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The hormone leptin plays a crucial role in maintenance of body weight and glucose homeostasis. This occurs through central and peripheral pathways, including regulation of insulin secretion by pancreatic beta cells. To study this further in mice, we disrupted the signaling domain of the leptin receptor gene in beta cells and hypothalamus. These mice develop obesity, fasting hyperinsulinemia, impaired glucose-stimulated insulin release, and glucose intolerance, similar to leptin receptor null mice. However, whereas complete loss of leptin function causes increased food intake, this tissue-specific attenuation of leptin signaling does not alter food intake or satiety responses to leptin. Moreover, unlike other obese models, these mice have reduced fasting blood glucose. These results indicate that leptin regulation of glucose homeostasis extends beyond insulin sensitivity to influence beta cell function, independent of pathways controlling food intake. These data suggest that defects in this adipoinsular axis could contribute to diabetes associated with obesity.
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Glucosa/metabolismo , Homeostasis , Células Secretoras de Insulina/metabolismo , Leptina/metabolismo , Animales , Ingestión de Alimentos , Femenino , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Leptina , Sensibilidad y Especificidad , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Glucagon-like peptide 1 (GLP-1) is a hormone that has received significant attention as a therapy for diabetes because of its ability to stimulate insulin biosynthesis and release and to promote growth and survival of insulin-producing beta cells. While GLP-1 is produced from the proglucagon precursor by means of prohormone convertase (PC) 1/3 activity in enteroendocrine L cells, the same precursor is differentially processed by PC2 in pancreatic islet alpha cells to release glucagon, leaving GLP-1 trapped within a larger fragment with no known function. We hypothesized that we could induce GLP-1 production directly within pancreatic islets by means of delivery of PC1/3 and, further, that this intervention would improve the viability and function of islets. Here, we show that adenovirus-mediated expression of PC1/3 in alpha cells increases islet GLP-1 secretion, resulting in improved glucose-stimulated insulin secretion and enhanced survival in response to cytokine treatment. PC1/3 expression in alpha cells also improved performance after islet transplantation in a mouse model of type 1 diabetes, possibly by enhancing nuclear Pdx1 and insulin content of islet beta cells. These results demonstrate a unique strategy for liberating GLP-1 from directly within the target organ and highlight the potential for up-regulating islet GLP-1 production as a means of treating diabetes.