RESUMEN
Impaired proinsulin-to-insulin processing in pancreatic ß-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3-8); nonetheless, the role of specific SL species in ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in ß-cell function and T2D-associated ß-cell failure.
Asunto(s)
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Proinsulina/genética , Proinsulina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Esfingolípidos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Homeostasis , Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismoRESUMEN
OBJECTIVE: Beta cell dysfunction and death are critical steps in the development of both type 1 and type 2 diabetes (T1D and T2D), but the underlying mechanisms are incompletely understood. Activation of the essential tumor suppressor and transcription factor P53 (also known as TP53 and Trp53 in mice) was linked to beta cell death in vitro and has been reported in several diabetes mouse models and beta cells of humans with T2D. In this article, we set out to determine the beta cell specific role of P53 in beta cell dysfunction, cell death and development of diabetes in vivo. METHODS: We generated beta cell specific P53 knockout (P53BKO) mice and used complementary genetic, dietary and pharmacological models of glucose intolerance, beta cell dysfunction and diabetes development to evaluate the functional role of P53 selectively in beta cells. We further analyzed the effect of P53 ablation on beta cell survival in isolated pancreatic islets exposed to diabetogenic stress inducers ex vivo by flow cytometry. RESULTS: Beta cell specific ablation of P53/Trp53 failed to ameliorate glucose tolerance, insulin secretion or to increase beta cell numbers in genetic, dietary and pharmacological models of diabetes. Additionally, loss of P53 in beta cells did not protect against streptozotocin (STZ) induced hyperglycemia and beta cell death, although STZ-induced activation of classical pro-apoptotic P53 target genes was significantly reduced in P53BKO mice. In contrast, Olaparib mediated PARP1 inhibition protected against acute ex vivo STZ-induced beta cell death and islet destruction. CONCLUSIONS: Our study reveals that ablation of P53 specifically in beta cells is unexpectedly unable to attenuate beta cell failure and death in vivo and ex vivo. While during development and progression of diabetes, P53 and P53-regulated pathways are activated, our study suggests that P53 signaling is not essential for loss of beta cells or beta cell dysfunction. P53 in other cell types and organs may predominantly regulate systemic glucose homeostasis.
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Ratones , Animales , Células Secretoras de Insulina/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Glucosa/metabolismoRESUMEN
Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Supervivencia Celular/genética , Células Secretoras de Insulina/patología , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Diabetes Mellitus/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ratas , Estreptozocina/toxicidad , Tunicamicina/toxicidadRESUMEN
FTY720 is a new oral immunomodulatory therapy for the treatment of multiple sclerosis (MS). There is strong evidence that FTY720 has direct effects on brain resident cells such as astrocytes acting via sphingosine1phosphate (S1P) receptors. In the present study, the mRNA expression of S1P receptors as well as selected cytokines, chemokines and growth factors were investigated in primary murine astrocytes under inflammatory conditions in the presence or absence of the phosphorylated form of FTY720 (FTY720P). Following stimulation with either the proinflammatory cytokine tumor necrosis factorα (TNFα) or with bacterial lipopolysaccharide, there was an increased expression of the receptors S1P1 and S1P3, the cytokines and chemokines interleukin (IL)1ß, chemokine (CCmotif) ligand 2 (CCL2), CCL20 and chemokine (CXCmotif) ligand 12 as well as the growth factors insulinlike growth factor1, ciliary neurotrophic factor and glial cell linederived neurotrophic factor (GDNF). FTY720P led to an increased expression of IL1ß and GDNF at distinct time points following costimulation with TNFα compared with TNFα treatment alone. However, the presence of FTY720P did not have any further significant effects on the expression of S1P receptors, cytokines or growth factors, suggesting that the regulation of these target genes in astrocytes is not likely to be a major mechanism underlying the effect of FTY720P in diseases such as MS.