RESUMO
BACKGROUND: Cognitive deficits, especially in attention, are common in persons with MS (PwMS) and are associated with clinically meaningful outcomes, such as work disability and lower quality of life (QOL). In this study, we aimed to determine whether Cogmed Working Memory Training (CWMT) improves attention/working memory in PwMS displaying impairment in these domains. METHODS: This single blind, randomized controlled, pilot study compared the effects of CWMT, a five-week evidenced-based computer-assisted training program that is supported by weekly meetings with a coach, to standard medical care (treatment as usual). We recruited PwMS from one MS center (London (ON) Canada), aged 18-64, with an Expanded Disability Status Scale (EDSS) score of ≤ 7.0, and a visual acuity (corrected) of at least 20/70. Potential subjects had to demonstrate impaired attention on at least two of three measures (Paced Auditory Serial Addition Test [PASAT], Symbol Digit Modalities Test [SDMT], and/or DKEFS Color-Word Interference Test); these measures also served as the primary study outcomes. Subjects were randomized to either the CWMT or treatment as usual. Secondary cognitive outcomes included other measures of attention, memory, as well as a self-reported cognitive function measure. Self-reported measures of mood (depression and anxiety), pain, and QOL were also included as other secondary outcomes. Subjects received assessments at baseline, post-treatment, and 6-month follow-up, or an equivalent time period for the treatment as usual group. The two groups were compared at baseline on background measures using independent samples t-tests, Chi-Square tests, and Mann-Whitney U tests. To analyze primary and secondary outcomes, a non-parametric approach was used due the small sample size and that many of our outcomes did not meet assumptions for parametric analyses. Friedman's test was conducted followed by post hoc pairwise comparisons within each group using Wilcoxon Signed-Rank tests with Bonferroni corrected post hoc contrasts, which allowed us to examine for differences between time points. RESULTS: Of 30 subjects, 15 were assigned to CWMT. Significant training effects were noted in 1 of 3 primary attentional outcomes (DKEFS Color-Word Interference Test), 2 of 3 secondary attentional outcomes (Letter-Number Sequencing, Digit Span), and 1 mood scale (Hospital Anxiety and Depression scale (HADS) - Depression Subscale), ps < .025. No significant changes were observed in the treatment as usual group. CONCLUSION: This pilot study demonstrates that cognitive training with CWMT has the potential to improve attention/working memory in PwMS, as well as a potential positive effect on mood, in PwMS. Further exploration of this intervention in PwMS with attention/working memory impairment is warranted.
Assuntos
Memória de Curto Prazo , Qualidade de Vida , Adolescente , Adulto , Atenção , Canadá , Cognição , Humanos , Aprendizagem , Londres , Pessoa de Meia-Idade , Projetos Piloto , Método Simples-Cego , Adulto JovemRESUMO
Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.
Assuntos
Autofagia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Jejum , Células Secretoras de Insulina/patologia , Lisossomos/metabolismo , Obesidade/complicações , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Beclina-1/genética , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Tipo 2/etiologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Camundongos , Camundongos Mutantes , Mitocôndrias/patologia , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Regulação para CimaRESUMO
Mammalian target of rapamycin (mTOR) is a serine and threonine protein kinase that regulates numerous cellular functions, in particular, the initiation of protein translation. mTOR-mediated phosphorylation of both the translational repressor eukaryotic initiation factor 4E binding protein-1 and p70 S6 kinase are early events that control the translation initiation process. Rapamycin, an inhibitor of mTOR, is a potent immunosuppressant due, in part, to its ability to interfere with T-cell activation at the level of translation, and it has gained a prominent role in preventing the development and progression of rejection in pancreatic islet transplant recipients. The characterization of the insulin signaling cascade that modulates mTOR in insulin-sensitive tissues has been a major focus of investigation. Recently, the ability of nutrients, in particular the branched-chain amino acid leucine, to activate mTOR independent of insulin by a process designated as nutrient signaling has been identified. The beta-cell expresses components of the insulin signaling cascade and utilizes the metabolism of nutrients to affect insulin secretion. These combined transduction processes make the beta-cell an unique cell to study metabolic and autocrine regulation of mTOR signaling. Our studies have described the ability of insulin and IGFs in concert with the nutrients leucine, glutamine, and glucose to modulate protein translation through mTOR in beta-cells. These findings suggest that mitochondria-derived factors, ATP in particular, may be responsible for nutrient signaling. The significance of these findings is that the optimization of mitochondrial function is not only important for insulin secretion but may significantly impact the growth and proliferation of beta-cells through these mTOR signaling pathways.
Assuntos
Comunicação Autócrina/fisiologia , Ilhotas Pancreáticas/enzimologia , Proteínas Quinases/metabolismo , Animais , Mamíferos , Serina-Treonina Quinases TORRESUMO
Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from mitogens and the nutrients, glucose and amino acids, to regulate cellular growth and proliferation. Previous findings demonstrated that glucose robustly activates mTOR in an amino acid-dependent manner in rodent and human islets. Furthermore, activation of mTOR by glucose significantly increases rodent islet DNA synthesis that is abolished by rapamycin. Glucagon-like peptide-1 (GLP-1) agonists, through the production of cAMP, have been shown to enhance glucose-dependent proinsulin biosynthesis and secretion and to stimulate cellular growth and proliferation. The objective of this study was to determine if the glucose-dependent and cAMP-mediated mechanism by which GLP-1 agonists enhance beta-cell growth and proliferation is mediated, in part, through mTOR. Our studies demonstrated that forskolin-generated cAMP resulted in activation of mTOR at basal glucose concentrations as assessed by phosphorylation of S6K1, a downstream effector of mTOR. Conversely, an adenylyl cyclase inhibitor partially blocked glucose-induced S6K1 phosphorylation. Furthermore, the GLP-1 receptor agonist, Exenatide, dose-dependently enhanced phosphorylation of S6K1 at an intermediate glucose concentration (8 mmol/l) in a rapamycin-sensitive manner. To determine the mechanism responsible for this potentiation of mTOR, the effects of intra- and extracellular Ca2+ were examined. Glyburide, an inhibitor of ATP-sensitive K+ channels (K(ATP) channels), provided partial activation of mTOR at basal glucose concentrations due to the influx of extracellular Ca2+, and diazoxide, an activator of KATP channels, resulted in partial inhibition of S6K1 phosphorylation by 20 mmol/l glucose. Furthermore, Exenatide or forskolin reversed the inhibition by diazoxide, probably through mobilization of intracellular Ca2+ stores by cAMP. BAPTA, a chelator of intracellular Ca2+, resulted in inhibition of glucose-stimulated S6K1 phosphorylation due to a reduction in cytosolic Ca2+ concentrations. Selective blockade of glucose-stimulated Ca2+ influx unmasked a protein kinase A (PKA)-sensitive component involved in the mobilization of intracellular Ca2+ stores, as revealed with the PKA inhibitor H-89. Overall, these studies support our hypothesis that incretin-derived cAMP participates in the metabolic activation of mTOR by mobilizing intracellular Ca2+ stores that upregulate mitochondrial dehydrogenases and result in enhanced ATP production. ATP can then modulate KATP channels, serve as a substrate for adenylyl cyclase, and possibly directly regulate mTOR activation.
Assuntos
Substâncias de Crescimento/fisiologia , Ilhotas Pancreáticas/fisiologia , Proteínas Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Colforsina/farmacologia , AMP Cíclico/farmacologia , Diazóxido/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição , Fosforilação , Serina-Treonina Quinases TORRESUMO
Our previous studies demonstrated that Wnt/GSK-3/ß-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human ß-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases ß-catenin nuclear translocation and ß-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human ß-cell proliferation while maintaining a ß-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis â¼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human ß-cell proliferation â¼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced ß-catenin-mediated and ß-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/ß-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human ß-cell proliferation while maintaining the ß-cell phenotype.
Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Via de Sinalização Wnt/fisiologia , Adulto , Amidas , Benzamidas , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Dioxóis , Perfilação da Expressão Gênica , Humanos , Piridinas , Receptor Cross-Talk/fisiologia , Trombospondinas/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Major bottlenecks in the expansion of human ß-cell mass are limited proliferation, loss of ß-cell phenotype, and increased apoptosis. In our previous studies, activation of Wnt and mTOR signaling significantly enhanced human ß-cell proliferation. However, isolated human islets displayed insulin signaling pathway resistance, due in part to chronic activation of mTOR/S6K1 signaling that results in negative feedback of the insulin signaling pathway and a loss of Akt phosphorylation and insulin content. We evaluated the effects of a new generation insulin sensitizer, MSDC-0160, on restoring insulin/IGF-1 sensitivity and insulin content in human ß-cells. This novel TZD has low affinity for binding and activation of PPARγ and has insulin-sensitizing effects in mouse models of diabetes and ability to lower glucose in Phase 2 clinical trials. MSDC-0160 treatment of human islets increased AMPK activity and reduced mTOR activity. This was associated with the restoration of IGF-1-induced phosphorylation of Akt, GSK-3, and increased protein expression of Pdx1. Furthermore, MSDC-0160 in combination with IGF-1 and 8 mM glucose increased ß-cell specific gene expression of insulin, pdx1, nkx6.1, and nkx2.2, and maintained insulin content without altering glucose-stimulated insulin secretion. Human islets were unable to simultaneously promote DNA synthesis and maintain the ß-cell phenotype. Lithium-induced GSK-3 inhibition that promotes DNA synthesis blocked the ability of MSDC-0160 to maintain the ß-cell phenotype. Conversely, MSDC-0160 prevented an increase in DNA synthesis by blocking ß-catenin nuclear translocation. Due to the counteracting pathways involved in these processes, we employed a sequential ex vivo strategy to first induce human islet DNA synthesis, followed by MSDC-0160 to promote the ß-cell phenotype and insulin content. This new generation PPARγ sparing insulin sensitizer may provide an initial tool for relieving inherent human islet insulin signaling pathway resistance that is necessary to preserve the ß-cell phenotype during ß-cell expansion for the treatment of diabetes.
Assuntos
Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/metabolismo , Piridinas/farmacologia , Tiazolidinedionas/farmacologia , Adenilato Quinase/metabolismo , Animais , Apoptose/genética , Núcleo Celular/metabolismo , Células Cultivadas , Replicação do DNA , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Humanos , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Fator de Crescimento Insulin-Like I/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas Nucleares , Fenótipo , Fosforilação , Pró-Proteína Convertase 2/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Técnicas de Cultura de Tecidos , Fatores de Transcrição , beta Catenina/metabolismoRESUMO
The aim of this study was to assess multifactorial ß-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of lipid droplets in ß-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in ß-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 µM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.
Assuntos
Ácidos Graxos não Esterificados/administração & dosagem , Glucose/administração & dosagem , Células Secretoras de Insulina/metabolismo , Animais , Western Blotting , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/ultraestrutura , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/metabolismo , Microscopia de Contraste de Fase , Complexos Multiproteicos , Perilipina-2 , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Triglicerídeos/metabolismoRESUMO
This perspective delineates approaches to develop therapeutic strategies to stimulate the proliferative potential of adult human ß-cells in vitro. Previous findings demonstrated that nutrients, through regulation of mTOR signaling, promote regenerative processes including DNA synthesis, cell cycle progression and ß-cell proliferation in rodent islets but rarely in human islets. Recently, we discovered that regulation of the Wnt/GSK-3/ß-catenin pathway by directly inhibiting GSK-3 with pharmacologic agents, in combination with nutrient activation of mTOR, was required to increase growth and proliferation in human islets. Studies also revealed that nuclear translocation of ß-catenin in response to GSK-3 inhibition regulated these processes and was rapamycin sensitive, indicating a role for mTOR. Human islets displayed a high level of insulin resistance consistent with the inability of exogenous insulin to activate Akt and engage the Wnt pathway by GSK-3 inhibition. This insulin resistance in human islets is not present in rodent islets and may explain the differential requirement in human islets to inhibit GSK-3 to enhance these regenerative processes. Human islets exhibited normal insulin secretion but a loss of insulin content, which was independent of all treatment conditions. The loss of insulin content may be related to insulin resistance, the isolation process or culture conditions. In this perspective, we provide strategies to enhance the proliferative capacity of adult human ß-cells and highlight important differences between human and rodent islets: the lack of a nutrient response, requirement for direct GSK-3 inhibition, insulin resistance and loss of insulin content that emphasize the physiological significance of conducting studies in human islets.
RESUMO
OBJECTIVE: Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/beta-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human beta-cell mass relevant to type 1 and 2 diabetes. RESEARCH DESIGN AND METHODS: Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of beta-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed. RESULTS: Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of beta-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human beta-cells. Nuclear translocation of beta-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin. CONCLUSIONS: A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human beta-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation.
Assuntos
Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Replicação do DNA , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Quinase 3 da Glicogênio Sintase/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Proteínas Quinases/metabolismo , Adolescente , Adulto , Idoso , Animais , Replicação do DNA/efeitos dos fármacos , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Insulina/farmacologia , Ilhotas Pancreáticas/enzimologia , Antígeno Ki-67/genética , Masculino , Pessoa de Meia-Idade , Proteínas Quinases/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR , Adulto JovemRESUMO
Obesity promotes insulin resistance and chronic inflammation. Disrupting any of several distinct steps in lipid synthesis decreases adiposity, but it is unclear if this approach coordinately corrects the environment that propagates metabolic disease. We tested the hypothesis that inactivation of FAS in the hypothalamus prevents diet-induced obesity and systemic inflammation. Ten weeks of high-fat feeding to mice with inactivation of FAS (FASKO) limited to the hypothalamus and pancreatic beta cells protected them from diet-induced obesity. Though high-fat fed FASKO mice had no beta-cell phenotype, they were hypophagic and hypermetabolic, and they had increased insulin sensitivity at the liver but not the periphery as demonstrated by hyperinsulinemic-euglycemic clamps, and biochemically by increased phosphorylated Akt, glycogen synthase kinase-3beta, and FOXO1 compared with wild-type mice. High-fat fed FASKO mice had decreased excretion of urinary isoprostanes, suggesting less oxidative stress and blunted tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) responses to endotoxin, suggesting less systemic inflammation. Pair-feeding studies demonstrated that these beneficial effects were dependent on central FAS disruption and not merely a consequence of decreased adiposity. Thus, inducing central FAS deficiency may be a valuable integrative strategy for treating several components of the metabolic syndrome, in part by correcting hepatic insulin resistance and suppressing inflammation.
Assuntos
Ácido Graxo Sintase Tipo I/deficiência , Hipotálamo/enzimologia , Inflamação/prevenção & controle , Obesidade/prevenção & controle , Animais , Gorduras na Dieta/administração & dosagem , Ácido Graxo Sintase Tipo I/genética , Fígado Gorduroso/enzimologia , Fígado Gorduroso/prevenção & controle , Feminino , Glucose/metabolismo , Homeostase , Técnicas In Vitro , Inflamação/enzimologia , Inflamação/etiologia , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Obesidade/enzimologia , Obesidade/etiologia , Estresse Oxidativo , Aumento de PesoRESUMO
OBJECTIVE: Low birth weight is associated with diabetes in adult life. Accelerated or "catch-up" postnatal growth in response to small birth size is thought to presage disease years later. Whether adult disease is caused by intrauterine beta-cell-specific programming or by altered metabolism associated with catch-up growth is unknown. RESEARCH DESIGN AND METHODS: We generated a new model of intrauterine growth restriction due to fatty acid synthase (FAS) haploinsufficiency (FAS deletion [FASDEL]). Developmental programming of diabetes in these mice was assessed from in utero to 1 year of age. RESULTS: FASDEL mice did not manifest catch-up growth or insulin resistance. beta-Cell mass and insulin secretion were strikingly increased in young FASDEL mice, but beta-cell failure and diabetes occurred with age. FASDEL beta-cells had altered proliferative and apoptotic responses to the common stress of a high-fat diet. This sequence appeared to be developmentally entrained because beta-cell mass was increased in utero in FASDEL mice and in another model of intrauterine growth restriction caused by ectopic expression of uncoupling protein-1. Increasing intrauterine growth in FASDEL mice by supplementing caloric intake of pregnant dams normalized beta-cell mass in utero. CONCLUSIONS: Decreased intrauterine body size, independent of postnatal growth and insulin resistance, appears to regulate beta-cell mass, suggesting that developing body size might represent a physiological signal that is integrated through the pancreatic beta-cell to establish a template for hyperfunction in early life and beta-cell failure with age.
Assuntos
Retardo do Crescimento Fetal/fisiopatologia , Peso Fetal/fisiologia , Células Secretoras de Insulina/fisiologia , Animais , Southern Blotting , Tamanho Corporal/genética , Tamanho Corporal/fisiologia , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Peso Fetal/genética , Técnica Clamp de Glucose , Immunoblotting , Imuno-Histoquímica , Insulina/metabolismo , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the K(ATP) channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of K(ATP) channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10-100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 microm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2-4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an approximately 62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.
Assuntos
Glucose/metabolismo , Ilhotas Pancreáticas/citologia , Canais de Potássio/química , Proteínas Quinases/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Ciclo Celular , Divisão Celular , Proliferação de Células , Células Cultivadas , Citosol/metabolismo , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Fase G2 , Glibureto/química , Glibureto/metabolismo , Masculino , Nifedipino/farmacologia , Potássio/química , Ratos , Ratos Sprague-Dawley , Fase S , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Timidina/metabolismoRESUMO
Lipid and glucose metabolism are adversely affected by diabetes, a disease characterized by pancreatic beta-cell dysfunction. To clarify the role of lipids in insulin secretion, we generated mice with beta-cell-specific overexpression (betaLPL-TG) or inactivation (betaLPL-KO) of lipoprotein lipase (LPL), a physiologic provider of fatty acids. LPL enzyme activity and triglyceride content were increased in betaLPL-TG islets; decreased LPL enzyme activity in betaLPL-KO islets did not affect islet triglyceride content. Surprisingly, both betaLPL-TG and betaLPL-KO mice were strikingly hyperglycemic during glucose tolerance testing. Impaired glucose tolerance in betaLPL-KO mice was present at one month of age, whereas betaLPL-TG mice did not develop defective glucose homeostasis until approximately five months of age. Glucose-simulated insulin secretion was impaired in islets isolated from both mouse models. Glucose oxidation, critical for ATP production and triggering of insulin secretion mediated by the ATP-sensitive potassium (KATP) channel, was decreased in betaLPL-TG islets but increased in betaLPL-KO islets. Islet ATP content was not decreased in either model. Insulin secretion was defective in both betaLPL-TG and betaLPL-KO islets under conditions causing calcium-dependent insulin secretion independent of the KATP channel. These results show that beta-cell-derived LPL has two physiologically relevant effects in islets, the inverse regulation of glucose metabolism and the independent mediation of insulin secretion through effects distal to membrane depolarization.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Lipase Lipoproteica/metabolismo , Animais , Membrana Celular/fisiologia , DNA Complementar , Glucose/farmacologia , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Lipase Lipoproteica/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Valores de Referência , Triglicerídeos/metabolismoRESUMO
Lipid accumulation in pancreatic beta-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the beta-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.