RESUMEN
KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.
Asunto(s)
Canal de Potasio KCNQ1 , Canales de Potasio con Entrada de Voltaje , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Simulación de Dinámica Molecular , Oocitos/metabolismo , Técnicas de Placa-ClampRESUMEN
The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5'-GGCTCG-3' in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT ) or complete ablation of Zbed6 leads to ~20-fold upregulation of the IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 upregulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of upregulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities, and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study adds new insights how the ZBED6-Igf2 axis affects muscle metabolism.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/metabolismo , Mioblastos/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular , Regulación de la Expresión Génica/genética , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , Mitocondrias/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Represoras/genética , Transducción de Señal/genética , Transcripción Genética/genética , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVE: To delineate potential mechanisms for fasting hyperglucagonemia in childhood obesity by studying the associations between fasting plasma glucagon concentrations and plasma lipid parameters and fat compartments. METHODS: Cross-sectional study of children and adolescents with obesity (n = 147) and lean controls (n = 43). Differences in free fatty acids (FFAs), triglycerides, insulin, and fat compartments (quantified by magnetic resonance imaging) across quartiles of fasting plasma glucagon concentration were analyzed. Differences in oral glucose tolerance test (OGTT) glucagon response was tested in high vs low FFAs, triglycerides, and insulin. Human islets of Langerhans were cultured at 5.5 mmol/L glucose and in the absence or presence of a FFA mixture with total FFA concentration of 0.5 mmol/L and glucagon secretion quantified. RESULTS: In children with obesity, the quartile with the highest fasting glucagon had higher insulin (201 ± 174 vs 83 ± 39 pmol/L, P < .01), FFAs (383 ± 52 vs 338 ± 109 µmol/L, P = .02), triglycerides (1.5 ± 0.9 vs 1.0 ± 0.7 mmol/L, P < .01), visceral adipose tissue volume (1.9 ± 0.8 vs 1.2 ± 0.3 dm3 , P < .001), and a higher prevalence of impaired glucose tolerance (IGT; 41% vs 8%, P = .01) than the lowest quartile. During OGTT, children with obesity and high insulin had a worse suppression of glucagon during the first 10 minutes after glucose intake. Glucagon secretion was 2.6-fold higher in islets treated with FFAs than in those not treated with FFAs. CONCLUSIONS: Hyperglucagonemia in childhood obesity is associated with hyperinsulinemia, high plasma FFAs, high plasma triglycerides, visceral adiposity, and IGT. The glucagonotropic effect of FFAs on isolated human islets provides a potential mechanism linking high fasting plasma FFAs and glucagon levels.
Asunto(s)
Adiposidad/fisiología , Ácidos Grasos no Esterificados/sangre , Glucagón/sangre , Intolerancia a la Glucosa/metabolismo , Grasa Intraabdominal/metabolismo , Obesidad Abdominal/metabolismo , Obesidad Infantil/metabolismo , Adolescente , Estudios de Casos y Controles , Células Cultivadas , Niño , Estudios de Cohortes , Estudios Transversales , Femenino , Glucagón/farmacología , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/complicaciones , Humanos , Grasa Intraabdominal/patología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Obesidad Abdominal/complicaciones , Obesidad Infantil/complicaciones , Regulación hacia ArribaRESUMEN
The naturally occurring quassinoid compound brusatol improves the survival of insulin-producing cells when exposed to the proinflammatory cytokines IL-1ß and IFN-γ in vitro. The aim of the present study was to investigate whether brusatol also promotes beneficial effects in mice fed a high-fat diet (HFD), and if so, to study the mechanisms by which brusatol acts. In vivo, we observed that the impaired glucose tolerance of HFD-fed male C57BL/6 mice was counteracted by a 2 wk treatment with brusatol. Brusatol treatment improved both ß-cell function and peripheral insulin sensitivity of HFD-fed mice. In vitro, brusatol inhibited ß-cell total protein and proinsulin biosynthesis, with an ED50 of â¼40 nM. In line with this, brusatol blocked cytokine-induced iNOS protein expression via inhibition of iNOS mRNA translation. Brusatol may have affected protein synthesis, at least in part, via inhibition of eukaryotic initiation factor 5A (eIF5A) hypusination, as eIF5A spermidine association and hypusination in RIN-5AH cells was reduced in a dose- and time-dependent manner. The eIF5A hypusination inhibitor GC7 promoted a similar effect. Both brusatol and GC7 protected rat RIN-5AH cells against cytokine-induced cell death. Brusatol reduced eIF5A hypusination and cytokine-induced cell death in EndoC-ßH1 cells as well. Finally, hypusinated eIF5A was reduced in vivo by brusatol in islet endocrine and endothelial islet cells of mice fed an HFD. The results of the present study suggest that brusatol improves glucose intolerance in mice fed an HFD, possibly by inhibiting protein biosynthesis and eIF5A hypusination.-Turpaev, K., Krizhanovskii, C., Wang, X., Sargsyan, E., Bergsten, P., Welsh, N. The protein synthesis inhibitor brusatol normalizes high-fat diet-induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Intolerancia a la Glucosa/tratamiento farmacológico , Factores de Iniciación de Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Cuassinas/farmacología , Proteínas de Unión al ARN/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Citocinas/metabolismo , Intolerancia a la Glucosa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiología , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Elevated levels of palmitate accentuate glucose-stimulated insulin secretion (GSIS) after short-term and cause beta-cell dysfunction after prolonged exposure. We investigated whether metformin, the first-line oral drug for treatment of T2DM, has beneficial effects on FFA-treated human islets and the potential mechanisms behind the effects. Insulin secretion, oxygen consumption rate (OCR), AMPK activation, endoplasmic reticulum (ER) stress and apoptosis were examined in isolated human islets after exposure to elevated levels of palmitate in the absence or presence of metformin. Palmitate exposure doubled GSIS after 2 days but halved after 7 days compared with control. Inclusion of metformin during palmitate exposure normalized insulin secretion both after 2 and 7 days. After 2-day exposure to palmitate, OCR and the marker of the adaptive arm of ER stress response (sorcin) were significantly raised, whereas AMPK phosphorylation, markers of pro-apoptotic arm of ER stress response (p-EIF2α and CHOP) and apoptosis (cleaved caspase 3) were not affected. Presence of metformin during 2-day palmitate exposure normalized OCR and sorcin levels. After 7-day exposure to palmitate, OCR and sorcin were not significantly different from control level, p-AMPK was reduced and p-EIF2α, CHOP and cleaved caspase 3 were strongly upregulated. Presence of metformin during 7-day culture with palmitate normalized the level of p-AMPK, p-EIF2α, CHOP and cleaved caspase 3 but significantly increased the level of sorcin. Our study demonstrates that metformin prevents early insulin hypersecretion and later decrease in insulin secretion from palmitate-treated human islets by utilizing different mechanisms.
Asunto(s)
Ácidos Grasos/farmacología , Hipoglucemiantes/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Metformina/farmacología , Apoptosis/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Insulina/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Técnicas de Cultivo de Órganos , Consumo de Oxígeno/efectos de los fármacos , Fosforilación/efectos de los fármacosRESUMEN
In obese children with high circulating concentrations of free fatty acid palmitate, we have observed that insulin levels at fasting and in response to a glucose challenge were several times higher than in obese children with low concentrations of the fatty acid as well as in lean controls. Declining and even insufficient insulin levels were observed in obese adolescents with high levels of the fatty acid. In isolated human islets exposed to palmitate we have observed insulin hypersecretion after 2 days exposure. In contrast, insulin secretion from the islets was reduced after 7 days culture in the presence of the fatty acid. This study aims at identifying islet-related biological events potentially linked with the observed insulin hypersecretion and later secretory decline in these obese children and adolescents using the islet model. We analyzed protein expression data obtained from human islets exposed to elevated palmitate levels for 2 and 7 days by an improved methodology for statistical analysis of differentially expressed proteins. Protein profiling of islet samples by liquid chromatography-tandem mass spectrometry identified 115 differentially expressed proteins (DEPs). Several DEPs including sorcin were associated with increased glucose-stimulated insulin secretion in islets after 2 days of exposure to palmitate. Similarly, several metabolic pathways including altered protein degradation, increased autophagy, altered redox condition, and hampered insulin processing were coupled to the functional impairment of islets after 7 days of culture in the presence of palmitate. Such biological events, once validated in the islets, may give rise to novel treatment strategies aiming at normalizing insulin levels in obese children with high palmitate levels, which may reduce or even prevent obesity-related type 2 diabetes mellitus.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Obesidad/genética , Ácido Palmítico/farmacología , Adolescente , Autofagia/efectos de los fármacos , Autofagia/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Niño , Cromatografía Liquida , Biología Computacional/métodos , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Ayuno , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Secreción de Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/patología , Ácido Palmítico/metabolismo , Proteolisis , Proteómica/métodos , Espectrometría de Masas en Tándem , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Long-term exposure to elevated levels of free fatty acids (FFAs) is deleterious for beta-cell function and may contribute to development of type 2 diabetes mellitus (T2DM). Whereas mechanisms of impaired glucose-stimulated insulin secretion (GSIS) in FFA-treated beta-cells have been intensively studied, biological events preceding the secretory failure, when GSIS is accentuated, are poorly investigated. To identify these early events, we performed genome-wide analysis of gene expression in isolated human islets exposed to fatty acid palmitate for different time periods. RESULTS: Palmitate-treated human islets showed decline in beta-cell function starting from day two. Affymetrix Human Transcriptome Array 2.0 identified 903 differentially expressed genes (DEGs). Mapping of the genes onto pathways using KEGG pathway enrichment analysis predicted four islet biology-related pathways enriched prior but not after the decline of islet function and three pathways enriched both prior and after the decline of islet function. DEGs from these pathways were analyzed at the transcript level. The results propose that in palmitate-treated human islets, at early time points, protective events, including up-regulation of metallothioneins, tRNA synthetases and fatty acid-metabolising proteins, dominate over deleterious events, including inhibition of fatty acid detoxification enzymes, which contributes to the enhanced GSIS. After prolonged exposure of islets to palmitate, the protective events are outweighed by the deleterious events, which leads to impaired GSIS. CONCLUSIONS: The study identifies temporal order between different cellular events, which either promote or protect from beta-cell failure. The sequence of these events should be considered when developing strategies for prevention and treatment of the disease.
Asunto(s)
Islotes Pancreáticos/efectos de los fármacos , Ácido Palmítico/farmacología , Adulto , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/fisiología , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Cultivo Primario de Células , Factores de Tiempo , Transcriptoma/efectos de los fármacosRESUMEN
Studies on the pathophysiology of type 2 diabetes mellitus (T2DM) have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our findings provide insight into how saturated fatty acids contribute to islet cell dysfunction by affecting the granule maturation process and confirmation in human islets of some previous findings from rodent islet and cell-line studies.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Palmitatos/farmacología , Proteómica , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Secreción de Insulina , Masculino , Persona de Mediana Edad , Proinsulina/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Free fatty acids (FFAs) acutely stimulate insulin secretion from pancreatic islets. Conflicting results have been presented regarding this effect at non-stimulatory glucose concentration, however. The aim of our study was to investigate how long-chain FFAs affect insulin secretion from isolated human pancreatic islets in the presence of physiologically fasting glucose concentrations and to explore the contribution of mitochondria to the effects on secretion. METHODS: Insulin secretion from human pancreatic islets was measured from short-term static incubation or perfusion system at fasting glucose concentration (5.5 mM) with or without 4 different FFAs (palmitate, palmitoleate, stearate, and oleate). The contribution of mitochondrial metabolism to the effects of fatty acid-stimulated insulin secretion was explored. RESULTS: The average increase in insulin secretion, measured from statically incubated and dynamically perifused human islets, was about 2-fold for saturated free fatty acids (SFAs) (palmitate and stearate) and 3-fold for mono-unsaturated free fatty acids (MUFAs) (palmitoleate and oleate) compared with 5.5 mmol/l glucose alone. Accordingly, MUFAs induced 50 % and SFAs 20 % higher levels of oxygen consumption compared with islets exposed to 5.5 mmol/l glucose alone. The effect was due to increased glycolysis. When glucose was omitted from the medium, addition of the FFAs did not affect oxygen consumption. However, the FFAs still stimulated insulin secretion from the islets although secretion was more than halved. The mitochondria-independent action was via fatty acid metabolism and FFAR1/GPR40 signaling. CONCLUSIONS: The findings suggest that long-chain FFAs acutely induce insulin secretion from human islets at physiologically fasting glucose concentrations, with MUFAs being more potent than SFAs, and that this effect is associated with increased glycolytic flux and mitochondrial respiration.
RESUMEN
Long-term exposure of beta cells to saturated fatty acids impairs insulin secretion and increases apoptosis. In contrast, unsaturated fatty acids protect beta-cells from the long-term negative effects of saturated fatty acids. We aimed to identify the mechanisms underlying this protective action of unsaturated fatty acids. To address the aim, insulin-secreting MIN6 cells were exposed to palmitate in the absence or presence of oleate and analyzed by using nano-LC MS/MS based proteomic approach. Important findings were validated by using alternative approaches. Proteomic analysis identified 34 proteins differentially expressed in the presence of palmitate compared to control samples. These proteins play a role in insulin processing, mitochondrial function, metabolism of biomolecules, calcium homeostasis, exocytosis, receptor signaling, ER protein folding, antioxidant activity and anti-apoptotic function. When oleate was also present during culture, expression of 15 proteins was different from the expression in the presence of palmitate alone. Most of the proteins affected by oleate are targets of the ER stress response and play a pro-survival role in beta cells such as protein folding and antioxidative defence. We conclude that restoration of pro-survival pathways of the ER stress response is a major mechanism underlying the protective effect of unsaturated fatty acids in beta-cells treated with saturated fatty acids.
Asunto(s)
Linfocitos B/metabolismo , Estrés del Retículo Endoplásmico/genética , Insulinoma/tratamiento farmacológico , Proteómica , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Línea Celular Tumoral , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Insulinoma/metabolismo , Insulinoma/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ácido Oléico/administración & dosificación , Ácido Oléico/metabolismo , Oxidación-Reducción , Palmitatos/administración & dosificación , Palmitatos/metabolismo , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: The prevalence of obesity-related diabetes in childhood is increasing and circulating levels of nonesterified fatty acids may constitute a link. Here, the association between palmitate and insulin secretion was investigated in vivo and in vitro. METHODS: Obese and lean children and adolescents (n = 80) were included. Palmitate was measured at fasting; insulin and glucose during an oral glucose tolerance test (OGTT). Human islets were cultured for 0 to 7 d in presence of 0.5 mmol/l palmitate. Glucose-stimulated insulin secretion (GSIS), insulin content and apoptosis were measured. RESULTS: Obese subjects had fasting palmitate levels between 0.10 and 0.33 mmol/l, with higher average levels compared to lean subjects. While obese children with elevated palmitate (>0.20 mmol/l) had accentuated insulin levels during OGTT, obese adolescents with high palmitate had delayed first-phase insulin response. In human islets exposed to palmitate for 2 d GSIS was twofold enhanced, but after 7 d attenuated. Intracellular insulin content decreased time-dependently in islets cultured in the presence of palmitate and cleaved caspase 3 increased. CONCLUSION: The rapid accentuated and delayed insulin secretory responses observed in obese children and adolescents, respectively, with high palmitate levels may reflect changes in islet secretory activity and integrity induced by extended exposure to the fatty acid.
Asunto(s)
Hiperinsulinismo/sangre , Células Secretoras de Insulina/citología , Palmitatos/sangre , Adolescente , Adulto , Anciano , Células Cultivadas , Niño , Preescolar , Estudios Transversales , Complicaciones de la Diabetes/sangre , Ácidos Grasos no Esterificados/química , Femenino , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Secreción de Insulina , Masculino , Persona de Mediana Edad , Obesidad/sangre , Obesidad/complicaciones , Obesidad Infantil , Factores de TiempoRESUMEN
Fatty acids affect insulin secretion via metabolism and FFAR1-mediated signaling. Recent reports indicate that these two pathways act synergistically. Still it remains unclear how they interrelate. Taking into account the key role of mitochondria in insulin secretion, we attempted to dissect the metabolic and FFAR1-mediated effects of fatty acids on mitochondrial function. One-hour culture of MIN6 cells with palmitate significantly enhanced mitochondrial respiration. Antagonism or silencing of FFAR1 prevented the palmitate-induced rise in respiration. On the other hand, in the absence of extracellular palmitate FFAR1 agonists caused a modest increase in respiration. Using an agonist of the M3 muscarinic acetylcholine receptor and PKC inhibitor we found that in the presence of the fatty acid mitochondrial respiration is regulated via Gαq protein-coupled receptor signaling. The increase in respiration in palmitate-treated cells was largely due to increased glucose utilization and oxidation. However, glucose utilization was not dependent on FFAR1 signaling. Collectively, these results indicate that mitochondrial respiration in palmitate-treated cells is enhanced via combined action of intracellular metabolism of the fatty acid and the Gαq-coupled FFAR1 signaling. Long-term palmitate exposure reduced ATP-coupling efficiency of mitochondria and deteriorated insulin secretion. The presence of the FFAR1 antagonist during culture did not improve ATP-coupling efficiency, however, it resulted in enhanced mitochondrial respiration and improved insulin secretion after culture. Taken together, our study demonstrates that during palmitate exposure, integrated actions of fatty acid metabolism and fatty acid-induced FFAR1 signaling on mitochondrial respiration underlie the synergistic action of the two pathways on insulin secretion.
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Insulina/metabolismo , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ácido Palmítico/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Secreción de Insulina , Ratones , Mitocondrias/metabolismo , Transducción de SeñalRESUMEN
Prolonged exposure to palmitate impairs ß-cell function and mass. One of the proposed mechanisms is alteration in ceramide (Cer) generation. In the present study, exposure to palmitate induced the level of palmitoyl transferase and Cer synthases, enzymes of the Cer de novo and salvage pathways, and doubled total Cer levels, which was associated with decreased insulin secretion and augmented apoptosis in MIN6 cells and human islets. By inhibiting enzymes of the pathways pharmacologically with myriocin (ISP-1) or fumonisin B1 or by small interfering RNA (siRNA), we showed that Cer(14:0), Cer(16:0), Cer(20:1), and Cer(24:0) species, generated by the salvage pathway, are linked to the harmful effect of palmitate on ß-cells. Oleate attenuates negative effects of palmitate on ß-cells. When oleate was included during culture of MIN6 cells with palmitate, the palmitate-induced up-regulation of the enzymes of the de novo and salvage pathways was prevented resulting in normalized levels of all Cer species except Cer(20:1). Our data suggest that enhanced Cer generation in response to elevated palmitate levels involves both de novo and salvage pathways. However, the negative effects of palmitate on ß-cells are attributed to generation of Cer species Cer(14:0), Cer(16:0), and Cer(24:0) via acylation of sphingosine.
Asunto(s)
Ceramidas/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Palmitatos/farmacología , Esfingosina/metabolismo , Acilación , Animales , Línea Celular , Ceramidas/química , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Ratones , Ácido OléicoRESUMEN
Free fatty acids (FFAs) have pleiotropic effects on the pancreatic ß-cell. Although acute exposure to FFAs stimulates glucose-stimulated insulin secretion (GSIS), prolonged exposure impairs GSIS and causes apoptosis. FFAs exert their effects both via intracellular metabolism and interaction with the FFA receptor 1 (FFAR1/GPR40). Here we studied the role of FFAR1 in acute and long-term effects of palmitate on GSIS and insulin content in isolated human islets by using the FFAR1 agonist TAK-875 and the antagonist ANT203. Acute palmitate exposure potentiated GSIS approximately 3-fold, whereas addition of the antagonist decreased this potentiation to approximately 2-fold. In the absence of palmitate, the agonist caused a 40% increase in GSIS. Treatment with palmitate for 7 days decreased GSIS to 70% and insulin content to 25% of control level. These negative effects of long-term exposure to palmitate were ameliorated by FFAR1 inhibition and further aggravated by additional stimulation of the receptor. In the absence of extracellularly applied palmitate, long-term treatment with the agonist caused a modest increase in GSIS. The protective effect of FFAR1 inhibition was verified by using FFAR1-deficient MIN6 cells. Improved ß-cell function by the antagonist was paralleled by the decreased apoptosis and lowered oxidation of palmitate, which may represent the potential mechanisms of protection. We conclude that FFAR1 in the pancreatic ß-cell plays a substantial role not only in acute potentiation of GSIS by palmitate but also in the negative long-term effects of palmitate on GSIS and insulin content.
Asunto(s)
Islotes Pancreáticos/efectos de los fármacos , Ácido Palmítico/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Benzofuranos/farmacología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glucosa/farmacología , Humanos , Hidrazonas/química , Hidrazonas/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Estructura Molecular , Ácido Palmítico/administración & dosificación , Pirimidinas/química , Pirimidinas/farmacología , ARN Interferente Pequeño , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Sulfonas/farmacologíaRESUMEN
BACKGROUND: Elevated levels of lipids are detrimental for beta-cell function and mass. One of the mechanisms of how fatty acids induce apoptosis is development of the unfolded protein response (UPR). It is still far from understood how fatty acids activate the UPR, however. METHODS: We examined how palmitate-induced activation of the UPR was affected by altering the metabolism of the fatty acid in insulin-secreting INS-1E and MIN6 cell lines and intact human islets. To increase oxidation, we used low glucose (5.5 mM) or AICAR; and to reduce oxidation, we used high glucose (25 mM) or etomoxir. UPR was measured after 3, 24 and 48 hours of palmitate treatment. RESULTS: Modulation of palmitate oxidation by either glucose or the pharmacological agents did not affect palmitate-induced UPR activation. CONCLUSION: Our finding suggests that other factors than oxidation of palmitate play a role in the activation of UPR in fatty acid-treated beta-cells.
RESUMEN
BACKGROUND: Prolonged elevated levels of lipids have negative effects on beta-cell function and mass (lipotoxicity). To what extent exposure to high glucose concentration is important in the harmful effects of lipids (glucolipotoxicity) has been debated. METHODS: We addressed beta-cell lipotoxicity by measuring apoptosis in isolated intact control human islets and insulin-secreting cell lines MIN6 and INS-1E cultured in the presence of palmitate and low (5.5 mM) or high (25 mM) glucose for 48 hours. RESULTS: In both cell lines and human islets palmitate induced apoptosis after culture at low glucose. Palmitate-induced apoptosis was not increased after culture at high compared to low glucose in human islets and MIN6 cells but glucose-induced rise in apoptosis was observed in INS-1E cells. The rise in apoptosis in INS-1E cells was partially reversed by inclusion of AMPK-agonist AICAR. When CPT1-inhibitor etomoxir was included during culture at low glucose palmitate-triggered apoptosis was accentuated both in the islets and the cell lines. Palmitate oxidation in human islets and the cell lines was comparable after culture at low glucose. At high glucose, palmitate oxidation was reduced by 30% in human islets and MIN6 cells but by 80% in INS-1E cells. In INS-1E cells, AICAR increased oxidation of palmitate. Presence of etomoxir at low glucose decreased palmitate oxidation both in the islets and the cell lines. CONCLUSIONS: In summary, lipotoxicity is evident not only in the presence of high but also low glucose concentrations. Additional effects of glucose are prominent in INS-1E but not in MIN6 cells and intact control human islets, which are able to efficiently oxidize fatty acids at high glucose and in this way avoid glucolipotoxicity.
Asunto(s)
Glucosa/farmacología , Islotes Pancreáticos/efectos de los fármacos , Palmitatos/toxicidad , Acetil-CoA Carboxilasa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Humanos , Islotes Pancreáticos/enzimología , Ratones , Oxidación-Reducción , Palmitatos/metabolismo , Ratas , Técnicas de Cultivo de TejidosRESUMEN
Elevated blood levels of glucose and lipids in individuals with type 2 diabetes mellitus have been observed to cause impairment of insulin secretion from pancreatic beta-cells. Chronic exposure to either of the circulating fatty acid oleate or palmitate has different effects on the beta-cell. Whereas palmitate causes functional impairment of the beta-cell and apoptosis, oleate has only minor negative effects on beta-cell function and mass. The aim of the present study was to delineate mechanisms by which the fatty acids affect the beta-cell differently. In particular, the aim was to identify beta-cell proteins exclusively regulated by palmitate. INS-1E cells were cultured for 24h in medium supplemented with palmitate or oleate. After culture, cells were lysed and subjected to two-dimensional gel electrophoresis. Proteins specifically regulated by palmitate were excised from the gel and identified by peptide mass fingerprinting using MALDI-TOF MS. Proteins exclusively regulated by palmitate were classified into proteins of carbohydrate or protein metabolism and Ca(2+) or mRNA binding proteins. The specific palmitate-induced down-regulation of enzymes of glycolysis, proteins of protein turnover and anti-apoptotic protein may contribute to explain the different effects exerted by palmitate and oleate on beta-cell function and mass.
Asunto(s)
Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Palmitatos/farmacología , Proteómica/métodos , Diabetes Mellitus Tipo 2/metabolismo , Electroforesis en Gel Bidimensional/métodos , Glucólisis , Humanos , Procesamiento de Imagen Asistido por Computador , Células Secretoras de Insulina/citología , Espectrometría de Masas/métodos , Modelos Biológicos , Ácido Oléico/química , Ácido Palmítico/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Effects of non-esterified fatty acids (FAs) are accentuated when applied together with elevated glucose through preferential use of glucose as fuel, which leads to decreased oxidation of FAs. We examined how over-expression of the mitochondrial FA transporter carnitine palmitoyltransferase 1 (CPT1) affects glucose-stimulated insulin secretion (GSIS), apoptosis and ER stress in INS-1E cells cultured in the presence of elevated levels of glucose and palmitate. INS-1E cells were infected with Tet-ON regulated adenovirus containing CPT1 and cultured for 48h in the presence of 0.5mM palmitate and 20mM glucose. Over-expressing CPT1 lowered basal insulin secretion in a dose-dependent manner thereby improving GSIS from INS-1E cells. Also, apoptosis was alleviated and ER-stress markers p-eIF2alpha and CHOP were decreased in cells over-expressing CPT1. We conclude that regulated over-expression of CPT1 is beneficial for glucolipotoxic beta-cells.
Asunto(s)
Carnitina O-Palmitoiltransferasa/biosíntesis , Ácidos Grasos no Esterificados/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Palmitatos/metabolismo , Adenoviridae , Animales , Apoptosis , Carnitina O-Palmitoiltransferasa/genética , Línea Celular , Retículo Endoplásmico/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Ácidos Grasos no Esterificados/toxicidad , Glucosa/toxicidad , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Palmitatos/farmacología , Ratas , Factor de Transcripción CHOP/metabolismoRESUMEN
Elevated levels of glucose and lipids are characteristics of individuals with type 2 diabetes mellitus (T2DM). The enhanced nutrient levels have been connected with deterioration of beta-cell function and impaired insulin secretion observed in these individuals. A strategy to improve beta-cell function in individuals with T2DM has been intermittent administration of K(ATP) channel openers. After such treatment, both the magnitude and kinetics of insulin secretion are markedly improved. In an attempt to further delineate mechanisms of how openers of K(ATP) channels improve beta-cell function, the effects of diazoxide on markers of endoplasmic reticulum (ER) stress was determined in beta-cells exposed to the fatty acid palmitate. The eukaryotic translation factor 2-alpha kinase 3 (EIF2AK3; also known as PERK) and endoplasmic reticulum to nucleus signaling 1 (ERN1; also known as IRE1) pathways, but not the activating transcription factor (ATF6) pathway of the unfolded protein response, are activated in such lipotoxic beta-cells. Inclusion of diazoxide during culture attenuated activation of the EIF2AK3 pathway but not the ERN1 pathway. This attenuation was associated with reduced levels of DNA-damage inducible transcript 3 (DDIT3; also known as CHOP) and beta-cell apoptosis was decreased. It is concluded that reduction of ER stress may be a mechanism by which diazoxide improves beta-cell function.
Asunto(s)
Diazóxido/farmacología , Retículo Endoplásmico/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Factor de Transcripción Activador 6/metabolismo , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Glucosa/farmacología , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Palmitatos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Conejos , Ratas , Factores de Transcripción del Factor Regulador X , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , eIF-2 Quinasa/metabolismoRESUMEN
Prolonged hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and apoptosis in insulin-producing beta-cells. The detrimental effects have been connected with glucose-induced lipid accumulation in the beta-cell. AMP-activated protein kinase (AMPK) agonist, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), promotes utilization of nutrient stores for energy production. It was tested how impaired GSIS and elevated apoptosis observed in insulinoma (INS)-1E cells after prolonged culture at 27 mM glucose were affected by the inclusion of 0.3 or 1 mM AICAR during culture. Glucose-induced impairment of insulin release was reverted by the inclusion of 0.3 but not 1 mM AICAR, which did not affect insulin content. The glucose-induced rise in triglyceride (TG) content observed in the cells cultured at 27 mM glucose was not altered by the inclusion of either 0.3 or 1 mM AICAR. Inclusion of 1 but not 0.3 mM AICAR during culture induced phosphorylation of AMPK and its downstream target acyl-CoA carboxylase. Phosphorylation was paralleled by reduced number of apoptotic cells and lowered expression of pro-apoptotic C/EBP homologous protein (CHOP). In conclusion, AICAR dose dependently improves beta-cell function and reduces apoptosis in beta-cells exposed to prolonged hyperglycaemia without changing TG levels.