RESUMO
AIMS/HYPOTHESIS: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. METHODS: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (ßS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. RESULTS: ßS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in ßS2KO islets. Islets isolated from ßS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of ßS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in ßS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. CONCLUSIONS/INTERPRETATION: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. DATA AVAILABILITY: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Camundongos , Humanos , Animais , Proinsulina/genética , Proinsulina/metabolismo , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Insulina/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
The transcriptional activity of Pdx1 is modulated by a diverse array of coregulatory factors that govern chromatin accessibility, histone modifications, and nucleosome distribution. We previously identified the Chd4 subunit of the nucleosome remodeling and deacetylase complex as a Pdx1-interacting factor. To identify how loss of Chd4 impacts glucose homeostasis and gene expression programs in ß-cells in vivo, we generated an inducible ß-cell-specific Chd4 knockout mouse model. Removal of Chd4 from mature islet ß-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature-to-mature insulin granules in Chd4-deficient ß-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation in vivo. RNA sequencing and assay for transposase-accessible chromatin with sequencing showed that lineage-labeled Chd4-deficient ß-cells have alterations in chromatin accessibility and altered expression of genes critical for ß-cell function, including MafA, Slc2a2, Chga, and Chgb. Knockdown of CHD4 from a human ß-cell line revealed similar defects in insulin secretion and alterations in several ß-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining ß-cell function. ARTICLE HIGHLIGHTS: Pdx1-Chd4 interactions were previously shown to be compromised in ß-cells from human donors with type 2 diabetes. ß-Cell-specific removal of Chd4 impairs insulin secretion and leads to glucose intolerance in mice. Expression of key ß-cell functional genes and chromatin accessibility are compromised in Chd4-deficient ß-cells. Chromatin remodeling activities enacted by Chd4 are essential for ß-cell function under normal physiological conditions.
Assuntos
Cromatina , Diabetes Mellitus Tipo 2 , Camundongos , Animais , Humanos , Cromatina/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Diabetes Mellitus Tipo 2/genética , DNA Helicases/genética , Camundongos Knockout , Expressão Gênica , GlucoseRESUMO
BACKGROUND: Stress responses within the ß cell have been linked with both increased ß cell death and accelerated immune activation in type 1 diabetes (T1D). At present, information on the timing and scope of these responses as well as disease-related changes in islet ß cell protein expression during T1D development is lacking. METHODS: Data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. FINDINGS: In islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in stress mitigation and maintenance of ß cell function, followed by loss of expression of protective proteins that heralded diabetes onset. EIF2 signalling and the unfolded protein response, mTOR signalling, mitochondrial function, and oxidative phosphorylation were commonly modulated pathways in both NOD mice and NOD-SCID mice rendered acutely diabetic by T cell adoptive transfer. Protein disulphide isomerase A1 (PDIA1) was upregulated in NOD islets and pancreatic sections from human organ donors with autoantibody positivity or T1D. Moreover, PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to non-diabetic controls. INTERPRETATION: We identified a core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of ß cell stress in T1D. FUNDING: NIH (R01DK093954, DK127308, U01DK127786, UC4DK104166, R01DK060581, R01GM118470, and 5T32DK101001-09). VA Merit Award I01BX001733. JDRF (2-SRA-2019-834-S-B, 2-SRA-2018-493-A-B, 3-PDF-20016-199-A-N, 5-CDA-2022-1176-A-N, and 3-PDF-2017-385-A-N).
Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Animais , Criança , Feminino , Humanos , Camundongos , Biomarcadores/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteômica , Células Secretoras de InsulinaRESUMO
A bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking nuclear matrix protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet-induced obesity, male wild-type and Nmp4-/- mice (8 weeks) were fed either low-fat diet or high-fat diet (HFD) for 12 weeks. Endpoint parameters included bone architecture, structural and estimated tissue-level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance, and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4-/- mice exhibited deficits in pancreatic ß-cell function and were modestly glucose intolerant under normal diet conditions. Despite the ß-cell deficits, the Nmp4-/- mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic ß-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Animais , Dieta Hiperlipídica/efeitos adversos , Humanos , Insulina , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas à Matriz Nuclear/metabolismo , Hormônio Paratireóideo , Fatores de Transcrição/metabolismoRESUMO
Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes. In this study, we characterized the metabolic phenotypes of the adult GIRKO mice acutely switched to high-fat diet (HFD) feeding in order to identify additional metabolic challenges required for disease progression. Distinct from other diet-induced obesity (DIO) and genetic models (e.g., db/db mice), GIRKO mice remained leaner on HFD feeding, but developed other cardinal features of insulin resistance syndrome. GIRKO mice rapidly developed hyperglycemia despite compensatory increases in ß-cell mass and hyperinsulinemia. Furthermore, GIRKO mice also had impaired oral glucose tolerance and a limited glucose-lowering benefit from exendin-4, suggesting that the blunted incretin effect contributed to hyperglycemia. Secondly, GIRKO mice manifested severe dyslipidemia while on HFD due to elevated hepatic lipid secretion, serum triglyceride concentration, and lipid droplet accumulation in hepatocytes. Thirdly, GIRKO mice on HFD had increased inflammatory cues in the gut, which were associated with the HFD-induced microbiome alterations and increased serum lipopolysaccharide (LPS). In conclusion, our studies identified important gene/diet interactions contributing to diabetes progression, which might be leveraged to develop more efficacious therapies.
Assuntos
Diabetes Mellitus Tipo 2 , Dieta Hiperlipídica , Intolerância à Glucose , Transportador de Glucose Tipo 4 , Hiperglicemia , Resistência à Insulina , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Transportador de Glucose Tipo 4/biossíntese , Transportador de Glucose Tipo 4/metabolismo , Hiperglicemia/sangue , Hiperglicemia/etiologia , Hiperglicemia/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Offspring of obese mothers suffer higher risks of type 2 diabetes due to increased adiposity and decreased ß cell function. To date, the sex-differences in offspring islet insulin secretion during early life has not been evaluated extensively, particularly prior to weaning at postnatal day 21 (P21). To determine the role of maternal obesity on offspring islet insulin secretion, C57BL/6J female dams were fed chow or western diet from 4 weeks prior to mating to induce maternal obesity. First, offspring of chow-fed and obese dams were evaluated on postnatal day 21 (P21) prior to weaning for body composition, glucose and insulin tolerance, and islet phasic insulin-secretion. Compared to same-sex controls, both male and female P21 offspring born to obese dams (MatOb) had higher body adiposity and exhibited sex-specific differences in glucose tolerance and insulin secretion. The male MatOb offspring developed the highest extent of glucose intolerance and lowest glucose-induced insulin secretion. In contrast, P21 female offspring of obese dams had unimpaired insulin secretion. Using SAX-HPLC, we found that male MatOb had a decrease in pancreatic heparan sulfate glycosaminoglycan, which is a macromolecule critical for islet health. Notably, 8-weeks-old offspring of obese dams continued to exhibit a similar pattern of sex-differences in glucose intolerance and decreased islet insulin secretion. Overall, our study suggests that maternal obesity induces sex-specific changes to pancreatic HSG in offspring and a lasting effect on offspring insulin secretion, leading to the sex-differences in glucose intolerance.
Assuntos
Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Obesidade Materna/metabolismo , Pâncreas/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Adiposidade , Animais , Dieta Hiperlipídica , Feminino , Glucose , Intolerância à Glucose/metabolismo , Intolerância à Glucose/fisiopatologia , Glicosaminoglicanos/efeitos adversos , Humanos , Secreção de Insulina , Masculino , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Fatores SexuaisRESUMO
Type 1 diabetes (T1D) is a consequence of autoimmune ß cell destruction, but the role of lipids in this process is unknown. We previously reported that activation of Ca2+-independent phospholipase A2ß (iPLA2ß) modulates polarization of macrophages (MΦ). Hydrolysis of the sn-2 substituent of glycerophospholipids by iPLA2ß can lead to the generation of oxidized lipids (eicosanoids), pro- and antiinflammatory, which can initiate and amplify immune responses triggering ß cell death. As MΦ are early triggers of immune responses in islets, we examined the impact of iPLA2ß-derived lipids (iDLs) in spontaneous-T1D prone nonobese diabetic mice (NOD), in the context of MΦ production and plasma abundances of eicosanoids and sphingolipids. We find that (a) MΦNOD exhibit a proinflammatory lipid landscape during the prediabetic phase; (b) early inhibition or genetic reduction of iPLA2ß reduces production of select proinflammatory lipids, promotes antiinflammatory MΦ phenotype, and reduces T1D incidence; (c) such lipid changes are reflected in NOD plasma during the prediabetic phase and at T1D onset; and (d) importantly, similar lipid signatures are evidenced in plasma of human subjects at high risk for developing T1D. These findings suggest that iDLs contribute to T1D onset and identify select lipids that could be targeted for therapeutics and, in conjunction with autoantibodies, serve as early biomarkers of pre-T1D.
Assuntos
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/etiologia , Metabolismo dos Lipídeos , Macrófagos Peritoneais/metabolismo , Adolescente , Animais , Criança , Diabetes Mellitus Tipo 1/terapia , Eicosanoides/metabolismo , Ácidos Graxos/metabolismo , Feminino , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Cetonas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Macrófagos Peritoneais/patologia , Macrófagos Peritoneais/transplante , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Naftalenos/farmacologiaRESUMO
The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic ß-cell has not been tested. We used an informatics-based approach to develop a transcriptional signature of ß-cell GA stress using existing RNA sequencing and microarray data sets generated using human islets from donors with diabetes and islets where type 1 (T1D) and type 2 (T2D) diabetes had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated. In parallel, we generated an RNA-sequencing data set from human islets treated with brefeldin A (BFA), a known GA stress inducer. Overlapping the T1D and T2D groups with the BFA data set, we identified 120 and 204 differentially expressed genes, respectively. In both the T1D and T2D models, pathway analyses revealed that the top pathways were associated with GA integrity, organization, and trafficking. Quantitative RT-PCR was used to validate a common signature of GA stress that included ATF3, ARF4, CREB3, and COG6 Taken together, these data indicate that GA-associated genes are dysregulated in diabetes and identify putative markers of ß-cell GA stress.
Assuntos
Simulação por Computador , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexo de Golgi/fisiologia , Humanos , Ilhotas Pancreáticas/metabolismo , Modelos Biológicos , Análise Serial de Proteínas , Estresse FisiológicoRESUMO
OBJECTIVES: Epidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic ß-cell function using rodent and in vitro models. METHODS: Beginning at 8 weeks of age, C57BL/6 J mice were concurrently fed a high-fat diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured INS-1 ß-cells and primary islets were exposed ex vivo to CS extract (CSE), and ß-cell function and viability were tested. Since CS increases ceramide accumulation in the lung and these bioactive sphingolipids have been implicated in pancreatic ß-cell dysfunction in diabetes, islet and ß-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry. RESULTS: Compared to HFD-fed, ambient air-exposed mice, HFD-fed and CS-exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and had accelerated worsening of their glucose tolerance. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of ß-cell dysfunction, significantly lower ß-cell mass (p = 0.017), reduced ß-cell proliferation (p = 0.006), and increased islet ceramide content compared to non-smoking control mice. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide levels, which was correlated with reduced insulin gene expression and glucose-stimulated insulin secretion, and increased ß-cell oxidative and endoplasmic reticulum (ER) stress. Treatment with the antioxidant N-acetylcysteine markedly attenuated the effects of CSE on ceramide levels, restored ß-cell function and survival, and increased cyclin D2 expression, while also reducing activation of ß-cell ER and oxidative stress. CONCLUSIONS: Our results indicate that CS exposure leads to impaired insulin production, processing, secretion and reduced ß-cell viability and proliferation. These effects were linked to increased ß-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of the mechanisms by which CS exposure impairs ß-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.
Assuntos
Ceramidas/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Fumar Tabaco/efeitos adversos , Animais , Glicemia/metabolismo , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Glucose/metabolismo , Teste de Tolerância a Glucose , Insulina/metabolismo , Resistência à Insulina/fisiologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicações , Obesidade/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Produtos do Tabaco/efeitos adversos , Aumento de PesoRESUMO
Alterations in endoplasmic reticulum (ER) calcium (Ca2+) levels diminish insulin secretion and reduce ß-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca2+ loss in ß cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP3R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca2+ imaging techniques in INS-1 ß cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP3R function. Our results revealed that the RyR is sensitive mainly to ER stress-induced dysfunction, whereas cytokine stress specifically alters IP3R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP3R with xestospongin C prevented ER Ca2+ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented ß-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin-treated INS-1 ß cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca2+ transients that depend on both ER Ca2+ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca2+ dynamics in ß cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca2+ may be an early pathogenic event in diabetes.
Assuntos
Sinalização do Cálcio , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células Secretoras de Insulina/patologia , Compostos Macrocíclicos/farmacologia , Masculino , Camundongos , Camundongos Mutantes , Oxazóis/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in ß-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by ß-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued ß-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve ß-cell health and function.
Assuntos
Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Linhagem Celular , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Ratos , Molécula 1 de Interação Estromal/genéticaRESUMO
PURPOSE OF REVIEW: Type 1 diabetes (T1D) is an autoimmune disease marked by ß-cell destruction. Immunotherapies for T1D have been investigated since the 1980s and have focused on restoration of tolerance, T cell or B cell inhibition, regulatory T cell (Treg) induction, suppression of innate immunity and inflammation, immune system reset, and islet transplantation. The purpose of this review is to provide an overview and lessons learned from single immunotherapy trials, describe recent and ongoing combination immunotherapy trials, and provide perspectives on strategies for future combination clinical interventions aimed at preserving insulin secretion in T1D. RECENT FINDINGS: Combination immunotherapies have had mixed results in improving short-term glycemic control and insulin secretion in recent-onset T1D. A handful of studies have successfully reached their primary end-point of improved insulin secretion in recent-onset T1D. However, long-term improvements glycemic control and the restoration of insulin independence remain elusive. Future interventions should focus on strategies that combine immunomodulation with efforts to alleviate ß-cell stress and address the formation of antigens that activate autoimmunity.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Imunoterapia , Animais , Ensaios Clínicos como Assunto , Humanos , Imunidade Inata , Insulina/metabolismo , Secreção de InsulinaRESUMO
Macrophages are important in innate and adaptive immunity. Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways. Activation of group VIA phospholipase A2 (iPLA2ß) causes accumulation of arachidonic acid, lysophospholipids, and eicosanoids that can promote inflammation and pathologic states. We examined the role of iPLA2ß in peritoneal macrophage immune function by comparing wild type (WT) and iPLA2ß-/- mouse macrophages. Compared with WT, iPLA2ß-/- macrophages exhibited reduced proinflammatory M1 markers when classically activated. In contrast, anti-inflammatory M2 markers were elevated under naïve conditions and induced to higher levels by alternative activation in iPLA2ß-/- macrophages compared with WT. Induction of eicosanoid (12-lipoxygenase (12-LO) and cyclooxygenase 2 (COX2))- and reactive oxygen species (NADPH oxidase 4 (NOX4))-generating enzymes by classical activation pathways was also blunted in iPLA2ß-/- macrophages compared with WT. The effects of inhibitors of iPLA2ß, COX2, or 12-LO to reduce M1 polarization were greater than those to enhance M2 polarization. Certain lipids (lysophosphatidylcholine, lysophosphatidic acid, and prostaglandin E2) recapitulated M1 phenotype in iPLA2ß-/- macrophages, but none tested promoted M2 phenotype. These findings suggest that (a) lipids generated by iPLA2ß and subsequently oxidized by cyclooxygenase and 12-LO favor macrophage inflammatory M1 polarization, and (b) the absence of iPLA2ß promotes macrophage M2 polarization. Reducing macrophage iPLA2ß activity and thereby attenuating macrophage M1 polarization might cause a shift from an inflammatory to a recovery/repair milieu.
Assuntos
Polaridade Celular , Fosfolipases A2 do Grupo VI/imunologia , Inflamação/enzimologia , Macrófagos/citologia , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Feminino , Fosfolipases A2 do Grupo VI/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/imunologiaRESUMO
Among the family of phospholipases A2 (PLA2s) are the Ca(2+)-independent PLA2s (iPLA2s) and they are designated group VI iPLA2s. In relation to secretory and cytosolic PLA2s, the iPLA2s are more recently described and details of their expression and roles in biological functions are rapidly emerging. The iPLA2s or patatin-like phospholipases (PNPLAs) are intracellular enzymes that do not require Ca(2+) for activity, and contain lipase (GXSXG) and nucleotide-binding (GXGXXG) consensus sequences. Though nine PNPLAs have been recognized, PNPLA8 (membrane-associated iPLA2γ) and PNPLA9 (cytosol-associated iPLA2ß) are the most widely studied and understood. The iPLA2s manifest a variety of activities in addition to phospholipase, are ubiquitously expressed, and participate in a multitude of biological processes, including fat catabolism, cell differentiation, maintenance of mitochondrial integrity, phospholipid remodeling, cell proliferation, signal transduction, and cell death. As might be expected, increased or decreased expression of iPLA2s can have profound effects on the metabolic state, CNS function, cardiovascular performance, and cell survival; therefore, dysregulation of iPLA2s can be a critical factor in the development of many diseases. This review is aimed at providing a general framework of the current understanding of the iPLA2s and discussion of the potential mechanisms of action of the iPLA2s and related involved lipid mediators.
Assuntos
Doenças do Sistema Nervoso Central/genética , Inflamação/genética , Neoplasias/genética , Fosfolipases A2 Independentes de Cálcio/metabolismo , Processamento Alternativo/genética , Motivos de Aminoácidos/genética , Cálcio/metabolismo , Doenças do Sistema Nervoso Central/patologia , Humanos , Inflamação/patologia , Lipase/genética , Lipase/metabolismo , Neoplasias/patologia , Fosfolipases A2 Independentes de Cálcio/genética , Transdução de SinaisRESUMO
Autoimmune ß-cell death leads to type 1 diabetes, and with findings that Ca(2+)-independent phospholipase A2ß (iPLA2ß) activation contributes to ß-cell death, we assessed the effects of iPLA2ß inhibition on diabetes development. Administration of FKGK18, a reversible iPLA2ß inhibitor, to NOD female mice significantly reduced diabetes incidence in association with 1) reduced insulitis, reflected by reductions in CD4(+) T cells and B cells; 2) improved glucose homeostasis; 3) higher circulating insulin; and 4) ß-cell preservation. Furthermore, FKGK18 inhibited production of tumor necrosis factor-α (TNF-α) from CD4(+) T cells and antibodies from B cells, suggesting modulation of immune cell responses by iPLA2ß-derived products. Consistent with this, 1) adoptive transfer of diabetes by CD4(+) T cells to immunodeficient and diabetes-resistant NOD.scid mice was mitigated by FKGK18 pretreatment and 2) TNF-α production from CD4(+) T cells was reduced by inhibitors of cyclooxygenase and 12-lipoxygenase, which metabolize arachidonic acid to generate bioactive inflammatory eicosanoids. However, adoptive transfer of diabetes was not prevented when mice were administered FKGK18-pretreated T cells or when FKGK18 administration was initiated with T-cell transfer. The present observations suggest that iPLA2ß-derived lipid signals modulate immune cell responses, raising the possibility that early inhibition of iPLA2ß may be beneficial in ameliorating autoimmune destruction of ß-cells and mitigating type 1 diabetes development.
Assuntos
Cálcio/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Animais , Linfócitos B , Disponibilidade Biológica , Linfócitos T CD4-Positivos , Diabetes Mellitus Tipo 1 , Feminino , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Fosfolipases A2 do Grupo VI/genética , Homeostase , Insulina/metabolismo , Células Secretoras de Insulina , Cetonas/química , Cetonas/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Estrutura Molecular , Naftalenos/química , Naftalenos/farmacologia , Isoformas de ProteínasRESUMO
Type 1 diabetes (T1D) results from autoimmune destruction of islet ß-cells, but the underlying mechanisms that contribute to this process are incompletely understood, especially the role of lipid signals generated by ß-cells. Proinflammatory cytokines induce ER stress in ß-cells and we previously found that the Ca(2+)-independent phospholipase A2ß (iPLA2ß) participates in ER stress-induced ß-cell apoptosis. In view of reports of elevated iPLA2ß in T1D, we examined if iPLA2ß participates in cytokine-mediated islet ß-cell apoptosis. We find that the proinflammatory cytokine combination IL-1ß+IFNγ, induces: a) ER stress, mSREBP-1, and iPLA2ß, b) lysophosphatidylcholine (LPC) generation, c) neutral sphingomyelinase-2 (NSMase2), d) ceramide accumulation, e) mitochondrial membrane decompensation, f) caspase-3 activation, and g) ß-cell apoptosis. The presence of a sterol regulatory element in the iPLA2ß gene raises the possibility that activation of SREBP-1 after proinflammatory cytokine exposure contributes to iPLA2ß induction. The IL-1ß+IFNγ-induced outcomes (b-g) are all inhibited by iPLA2ß inactivation, suggesting that iPLA2ß-derived lipid signals contribute to consequential islet ß-cell death. Consistent with this possibility, ER stress and ß-cell apoptosis induced by proinflammatory cytokines are exacerbated in islets from RIP-iPLA2ß-Tg mice and blunted in islets from iPLA2ß-KO mice. These observations suggest that iPLA2ß-mediated events participate in amplifying ß-cell apoptosis due to proinflammatory cytokines and also that iPLA2ß activation may have a reciprocal impact on ER stress development. They raise the possibility that iPLA2ß inhibition, leading to ameliorations in ER stress, apoptosis, and immune responses resulting from LPC-stimulated immune cell chemotaxis, may be beneficial in preserving ß-cell mass and delaying/preventing T1D evolution.
Assuntos
Apoptose , Citocinas/imunologia , Diabetes Mellitus Tipo 1/enzimologia , Fosfolipases A2 do Grupo VI/imunologia , Interferon gama/imunologia , Interleucina-1beta/imunologia , Ilhotas Pancreáticas/citologia , Adulto , Animais , Citocinas/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/fisiopatologia , Estresse do Retículo Endoplasmático , Feminino , Fosfolipases A2 do Grupo VI/genética , Humanos , Interferon gama/genética , Interleucina-1beta/genética , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Knockout , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/imunologiaRESUMO
Ongoing studies suggest an important role for iPLA2ß in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA2ßinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA2ß in biological processes. While BEL is recognized as a more potent inhibitor of iPLA2 than of cPLA2 or sPLA2, leading to its designation as a "specific" inhibitor of iPLA2, it has been shown to also inhibit non-PLA2 enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA2ß and membrane-associated iPLA2γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA2ß. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA2ß with a greater potency (100-fold) than iPLA2γ, (b) inhibition of iPLA2ß is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA2ß activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA2ß. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA2ß role in biological functions.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/prevenção & controle , Fosfolipases A2 do Grupo VI/antagonistas & inibidores , Células Secretoras de Insulina/patologia , Cetonas/farmacologia , Naftalenos/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Diabetes Mellitus/patologia , Dinoprostona/biossíntese , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glucose/farmacologia , Fosfolipases A2 do Grupo VI/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Cetonas/química , Cetonas/uso terapêutico , Camundongos , Miocárdio/metabolismo , Naftalenos/química , Naftalenos/uso terapêutico , Pironas/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Fatores de TempoRESUMO
ß-cell apoptosis is a significant contributor to ß-cell dysfunction in diabetes and ER stress is among the factors that contributes to ß-cell death. We previously identified that the Ca²âº-independent phospholipase A2ß (iPLA2ß), which in islets is localized in ß-cells, participates in ER stress-induced ß-cell apoptosis. Here, direct assessment of iPLA2ß role was made using ß-cell-specific iPLA2ß overexpressing (RIP-iPLA2ß-Tg) and globally iPLA2ß-deficient (iPLA2ß-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2ß and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2ß, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and ß-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, ß-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2ß deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2ß impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in ß-cells and (2) both over- or under-expression of iPLA2ß is deleterious to the ß-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2ß in islet ß-cells. These findings support the hypothesis that iPLA2ß induction under stress, as in diabetes, is a key component to amplifying ß-cell death processes.
Assuntos
Apoptose , Autofagia , Estresse do Retículo Endoplasmático , Regulação Enzimológica da Expressão Gênica , Fosfolipases A2 do Grupo IV/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Ceramidas/metabolismo , Diabetes Mellitus/enzimologia , Diabetes Mellitus/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/biossíntese , Fosfolipases A2 do Grupo IV/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Esfingomielina Fosfodiesterase/biossíntese , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Técnicas de Cultura de Tecidos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Type 1 Diabetes is characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing ß-cells in the pancreatic islets. Akt1/Protein Kinase B is the direct downstream target of PI3 Kinase activation, and has shown potent anti-apoptotic and proliferation-inducing activities. This study was designed to explore whether gene transfer of constitutively active Akt1 (CA-Akt1) would promote ß-cell survival and proliferation, thus be protective against experimental diabetes. In the study, a fiber-modified infectivity-enhanced adenoviral vector, Ad5RGDpK7, was used to deliver rat insulin promoter (RIP)-driven CA-Akt1 into ß-cells. Our data showed this vector efficiently delivered CA-Akt1 into freshly isolated pancreatic islets, and promoted islet cell survival and ß-cell proliferation in vitro. The therapeutic effect of the vector in vivo was assessed using streptozotocin (STZ)-induced diabetes mice. Two means of vector administration were explored: intravenous and intra-bile ductal injections. While direct vector administration into pancreas via bile-ductal injection resulted in local adverse effect, intravenous injection of the vectors offered therapeutic benefits. Further analysis suggests systemic vector administration caused endogenous Akt expression and activation in islets, which may be responsible, at least in part, for the protective effect of the infectivity-enhanced CA-Akt1 gene delivery vector. Taken together, our data suggest CA-Akt1 is effective in promoting ß-cell survival and proliferation in vitro, but direct in vivo use is compromised by the efficacy of transgene delivery into ß-cells. Nonetheless, the vector evoked the expression and activation of endogenous Akt in the islets, thus offering beneficial bystander effect against STZ-induced diabetes.
Assuntos
Adenoviridae/genética , Diabetes Mellitus Tipo 1/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células Secretoras de Insulina/citologia , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Processos de Crescimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Experimental/virologia , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The non-ß endocrine cells in pancreatic islets play an essential counterpart and regulatory role to the insulin-producing ß-cells in the regulation of blood-glucose homeostasis. While significant progress has been made towards the understanding of ß-cell regeneration in adults, very little is known about the regeneration of the non-ß endocrine cells such as glucagon-producing α-cells and somatostatin producing δ-cells. Previous studies have noted the increase of α-cell composition in diabetes patients and in animal models. It is thus our hypothesis that non-ß-cells such as α-cells and δ-cells in adults can regenerate, and that the regeneration accelerates in diabetic conditions. To test this hypothesis, we examined islet cell composition in a streptozotocin (STZ)-induced diabetes mouse model in detail. Our data showed the number of α-cells in each islet increased following STZ-mediated ß-cell destruction, peaked at Day 6, which was about 3 times that of normal islets. In addition, we found δ-cell numbers doubled by Day 6 following STZ treatment. These data suggest α- and δ-cell regeneration occurred rapidly following a single diabetes-inducing dose of STZ in mice. Using in vivo BrdU labeling techniques, we demonstrated α- and δ-cell regeneration involved cell proliferation. Co-staining of the islets with the proliferating cell marker Ki67 showed α- and δ-cells could replicate, suggesting self-duplication played a role in their regeneration. Furthermore, Pdx1(+)/Insulin(-) cells were detected following STZ treatment, indicating the involvement of endocrine progenitor cells in the regeneration of these non-ß cells. This is further confirmed by the detection of Pdx1(+)/glucagon(+) cells and Pdx1(+)/somatostatin(+) cells following STZ treatment. Taken together, our study demonstrated adult α- and δ-cells could regenerate, and both self-duplication and regeneration from endocrine precursor cells were involved in their regeneration.