RESUMO
OBJECTIVES: Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such as metformin, sulfonylureas, and glucagon-like peptide-1 (GLP) receptor agonist, there is growing interest in finding alternative methods to noninvasively treat this disease. It has been previously shown that low-intensity ultrasound stimulation of pancreatic ß-cells in mice can elicit insulin secretion as a potential treatment for this disease. This is desirable as therapeutic ultrasound has the ability to induce bioeffects while selectively focusing deep within tissues, allowing for modulation of hormone secretion in the pancreas to mitigate insufficient levels of insulin. METHODS: Exactly 800 kHz ultrasound with intensity 0.5 W/cm2 was administered 5 minutes continuously, that is, 100% duty cycle, to donor pancreatic human islets, followed by 1 hour incubation and RT-qPCR to assess the effect of ultrasound stimulation on gene expression. The genes were insulin (INS), glucagon (Glu), amylin (Amy), and binding immunoglobulin protein (BiP). Nine donor pancreatic human islets were used to assess insulin and glucagon secretion, while eight samples were used for amylin and BiP. Fold change (FC) was calculated to analyze the effect of ultrasound stimulation on the gene expression of the donor islet cells. High-glucose and thapsigargin-treated islets were utilized as positive controls. Cell viability testing was done using a Trypan Blue Exclusion Test. RESULTS: Ultrasound stimulation did not cause a statistically significant upregulation in any of the tested genes (INS FC = 1.15, P-value = .5692; Glu FC = 1.60, P-value = .2231; Amy FC, P-value = .2863; BiP FC = 2.68, P-value = .3907). CONCLUSIONS: The results of this study show that the proposed ultrasound treatment parameters do not appear to significantly affect gene expression of any gene tested.
Assuntos
Insulina , Ilhotas Pancreáticas , Terapia por Ultrassom , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Terapia por Ultrassom/métodos , Glucagon , Expressão Gênica/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologiaRESUMO
Here, we investigated transcriptional and trafficking mechanisms of human islet amyloid polypeptide (hIAPP) in normal and stressed ß-cells. In high glucose-challenged human islets and rat insulinoma cells overexpressing hIAPP, cell fractionation studies revealed increased accumulation of hIAPP. Unexpectedly, a significant fraction (up to 22%) of hIAPP was found in the nuclear soluble and chromatin-enriched fractions of cultured human islet and rat insulinoma cells. The nucleolar accumulation of monomeric forms of hIAPP did not have any adverse effect on the proliferation of ß-cells nor did it affect nucleolar organization or function. However, intact nucleolar organization and function were essential for hIAPP expression under normal and ER-stress conditions as RNA polymerase II inhibitor, α-amanitin, reduced hIAPP protein expression evoked by high glucose and thapsigargin. Promoter activity studies revealed the essential role of transcription factor FoxA2 in hIAPP promoter activation in ER-stressed ß-cells. Transcriptome and secretory studies demonstrate that the biosynthetic and secretory capacity of islet ß-cells was preserved during ER stress. Thus, the main reason for increased intracellular hIAPP accumulation is its enhanced biosynthesis under these adverse conditions.
Assuntos
Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Glucose/farmacologia , Fator 3-beta Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , RNA Polimerase II/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Fator 3-beta Nuclear de Hepatócito/genética , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , RNA Polimerase II/genética , Ratos , EdulcorantesRESUMO
Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet ß-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of ß-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP-membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Pâncreas/patologia , Agregação Patológica de Proteínas/patologia , Animais , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/análise , Pâncreas/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Dobramento de Proteína , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologiaRESUMO
OBJECTIVES: Our previously published studies showed the potential of therapeutic ultrasound (US) as a novel non-pharmacological alternative for the treatment of secretory deficiencies in type 2 diabetes. Despite showing enhanced insulin release from beta cells, these studies did not explore the potential effects of US treatment on other cells in the islets of Langerhans such as glucagon-secreting alpha cells or acinar cells of the exocrine pancreas. METHODS: We applied US parameters found capable of safely stimulating insulin secretion from pancreatic beta cells (f = 800 kHz, ISPTA = 0.5-1 W/cm2 , 5 minutes) to a diced rabbit pancreas model in culture plates (n = 6 per group). Released quantities of insulin and glucagon in response to US treatment were measured by collecting aliquots of the extracellular medium prior to the start of the treatment (t = 0 minute), immediately after treatment (t = 5 minutes) and 30 minutes after the end of treatment (t = 35 minutes). Potential release of digestive enzyme alpha-amylase as a result of US treatment was evaluated in rabbit pancreas experiments. Preliminary studies were also performed in a small number of human pancreatic islets in culture plates (n = 3 per group). The general integrity of the US-treated rabbit pancreatic tissue and human pancreatic islets was evaluated through histological analysis. RESULTS: While sham-treated rabbit pancreas samples showed decreased extracellular insulin content, there was an increase in insulin release at t = 5 minutes from samples treated with US at 800 kHz and 1 W/cm2 (P <.005). Furthermore, no further insulin release was detected at t = 35 minutes. No statistically significant difference in extracellular glucagon and alpha-amylase concentrations was observed between US-treated and sham rabbit pancreas groups. Preliminary studies in human islets appeared to follow trends observed in rabbit pancreas studies. Islet and other pancreatic tissue integrity did not appear to be affected by the US treatment. CONCLUSION: A potential US-based strategy for enhanced insulin release would require optimization of insulin secretion from pancreatic beta cells while minimizing glucagon and pancreatic enzyme secretions.
Assuntos
Diabetes Mellitus Tipo 2 , Glucagon , Animais , Insulina , Pâncreas/diagnóstico por imagem , Coelhos , alfa-AmilasesRESUMO
Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed ß-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed ß-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma ß-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in ß-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/patologia , Regulação para Baixo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Células Secretoras de Insulina/enzimologia , Insulinoma/enzimologia , Insulinoma/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Leupeptinas/farmacologia , Camundongos , Oligopeptídeos/farmacologia , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , RatosRESUMO
Human islet amyloid polypeptide or amylin (hA) is a 37-amino acid peptide hormone produced and co-secreted with insulin by pancreatic ß-cells. Under physiological conditions, hA regulates a broad range of biological processes including insulin release and slowing of gastric emptying, thereby maintaining glucose homeostasis. However, under the pathological conditions associated with type 2 diabetes mellitus (T2DM), hA undergoes a conformational transition from soluble random coil monomers to alpha-helical oligomers and insoluble ß-sheet amyloid fibrils or amyloid plaques. There is a positive correlation between hA oligomerization/aggregation, hA toxicity, and diabetes progression. Because the homeostatic balance between hA synthesis, release, and uptake is lost in diabetics and hA aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies investigating molecular mechanisms of hA uptake, trafficking, and degradation in pancreatic cells and its relevance to h's toxicity. We will also discuss the regulatory role of endocytosis and proteolytic pathways in clearance of toxic hA species. Finally, we will discuss potential pharmacological approaches for specific targeting of hA trafficking pathways and toxicity in islet ß-cells as potential new avenues toward treatments of T2DM patients.
Assuntos
Amiloidose/etiologia , Diabetes Mellitus Tipo 2/etiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/fisiologia , Ilhotas Pancreáticas/metabolismo , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Endocitose , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Complexo de Endopeptidases do Proteassoma/fisiologia , Agregados ProteicosRESUMO
Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin's clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma ß-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, keeping the net cytosolic amylin content low. An increase in hA accumulation in the nucleus of pancreatic cells correlated with its cytotoxicity, suggesting that its excessive accumulation in the nucleus is detrimental. hA interacted with 20S core and 19S lid subunits of the ß-cell proteasomal complex, as suggested by immunoprecipitation and confocal microscopy studies, which subsequently resulted in a decrease in the proteasome's proteolytic activity in these cells. In vitro binding and activity assays confirmed an intrinsic and potent ability of amylin to interact with the 20S core complex thereby modulating its proteolytic activity. Interestingly, less toxic and aggregation incapable rat amylin (rA) showed a comparable inhibitory effect on proteasome activity and protein ubiquitination, decoupling amylin aggregation/ toxicity and amylin-induced protein stress. In agreement with these studies, inhibition of proteasomal proteolytic activity significantly increased intracellular amylin content and toxicity. Taken together, our results suggest a pivotal role of proteasomes in amylin's turnover and detoxification in pancreatic cells.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Microscopia Confocal , Pâncreas/citologia , RatosRESUMO
Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the ß-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of ß-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of ß-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.
Assuntos
Cobre/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Agregados Proteicos , Multimerização Proteica , Sequência de Aminoácidos , Cátions Bivalentes/metabolismo , Humanos , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Amyloidosis is a biological event in which proteins undergo structural transitions from soluble monomers and oligomers to insoluble fibrillar aggregates that are often toxic to cells. Exactly how amyloid proteins, such as the pancreatic hormone amylin, aggregate and kill cells is still unclear. Islet amyloid polypeptide, or amylin, is a recently discovered hormone that is stored and co-released with insulin from pancreatic islet ß-cells. The pathology of type 2 diabetes mellitus (T2DM) is characterized by an excessive extracellular and intracellular accumulation of toxic amylin species, soluble oligomers and insoluble fibrils, in islets, eventually leading to ß-cell loss. Obesity and elevated serum cholesterol levels are additional risk factors implicated in the development of T2DM. Because the homeostatic balance between cholesterol synthesis and uptake is lost in diabetics, and amylin aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies exploring molecular mechanisms by which cholesterol and phospholipids modulate secondary structure, folding and aggregation of human amylin and other amyloid proteins on membranes and in cells. Amylin turnover and toxicity in pancreatic cells and the regulatory role of cholesterol in these processes are also discussed.
Assuntos
Amiloidose/fisiopatologia , Colesterol/fisiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/fisiopatologia , Fosfolipídeos/fisiologia , Dobramento de Proteína , Sequência de Aminoácidos , Amiloidose/etiologia , Animais , Diabetes Mellitus Tipo 2/fisiopatologia , Humanos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Human amylin-derived oligomers and aggregates are believed to play an important role in the pathogenesis of type II diabetes mellitus (T2DM). In addition to amylin-evoked cell attrition, T2DM is often accompanied by elevated serum copper levels. Although previous studies have shown that human amylin, in the course of its aggregation, produces hydrogen peroxide (H2O2) in solution, and that this process is exacerbated in the presence of copper(ii) ions (Cu(2+)), very little is known about the mechanism of interaction between Cu(2+) and amylin in pancreatic ß-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu(2+) and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu(2+) on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) ß-cells. Our results indicate that Cu(2+) interacts with human and rat amylin to form metalo-peptide complexes with low aggregative and oxidative properties. Human and non-amyloidogenic rat amylin produced minute (nM) amounts of H2O2, the accumulation of which was slightly enhanced in the presence of Cu(2+). In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu(2+) produced µM concentrations of H2O2 surpassing the amylin effect by several fold. The current study shows that human and rat amylin not only produce but also quench H2O2, and that human but not rat amylin significantly decreases the amount of H2O2 in solution produced by Cu(2+) and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu(2+) and glutathione. Furthermore, Cu(2+) mitigated the toxic effect of human amylin by inhibiting activation of pro-apoptotic caspase-3 and stress-kinase signaling pathways in rat pancreatic insulinoma cells in part by stabilizing human amylin in its native conformational state. This sacrificial quenching of metal-catalyzed ROS by human amylin and copper's anti-aggregative and anti-apoptotic properties suggest a novel and protective role for the copper-amylin complex.
Assuntos
Cobre/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dicroísmo Circular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Glutationa/química , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Radical Hidroxila/toxicidade , Íons/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RatosRESUMO
Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet ß-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect ß-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Multimerização Proteica , Animais , Linhagem Celular Tumoral , Membrana Celular/patologia , Clatrina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Endocitose , Humanos , Células Secretoras de Insulina/patologia , RatosRESUMO
Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.
Assuntos
Complexo de Golgi/ultraestrutura , Imageamento Tridimensional/métodos , Vesículas Secretórias/ultraestrutura , Células Acinares/diagnóstico por imagem , Células Acinares/metabolismo , Animais , Membrana Celular/ultraestrutura , Camundongos , Camundongos Endogâmicos ICR , Microscopia de Força Atômica , Microscopia Eletrônica , Biogênese de Organelas , Relação Estrutura-Atividade , UltrassonografiaRESUMO
To the best of our knowledge, therapeutic ultrasound (TUS) is thus far an unexplored means of delivering mechanical stimulation to cardiomyocyte cultures, which is necessary to engineer a more mature cardiomyocyte phenotype in vitro. Spectral ultrasound (SUS) may provide a way to non-invasively, non-disruptively and inexpensively monitor growth and change in cell cultures over long periods. Compared with other measurement methods, SUS as an acoustic measurement tool will not be affected by an acoustic therapy, unlike electrical measurement methods, in which motion caused by acoustic therapy can affect measurements. Further SUS has the potential to provide functional as well as morphological information in cell cultures. Human induced pluripotent stem cell cardiomyocytes (iPS-CMs) were imaged with calcium fluorescence microscopy while TUS was being applied. TUS was applied at 600 kHz and 1, 3.4 and 6 W/cm2 for a continuous 1 s pulse. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. At 600 kHz, TUS at 1 and 6 W/cm2 had significant effects on the shortening of both the repolarization rate and instantaneous beat rate of the iPS-CMs (p < 0.05), while TUS at 3.4 and 6 W/cm2 had significant effects on the shortening of the calcium spike amplitude (p < 0.05). Three SUS measures and one gray-level measure were captured from the iPS-CM monolayers while they were simultaneously being imaged with calcium-labeled confocal microscopy. The gray-level measure performed the best of all SUS measures; however, it was not reliable enough to produce a consistent determination of the beat rate of the cell. Finally, SUS measures were captured using three different transducers while simultaneously applying TUS. A center-of-mass (COM) measure calculated from the wavelet transform scalogram of the time-averaged radiofrequency data revealed that SUS was able to detect a change in the frequency content of the reflected ultrasound at 1 and 6 W/cm2 before and after ultrasound application (p < 0.05), showing promise for the ability of SUS to measure changes in the beating behavior of iPS-CMs. Overall, SUS is promising as a method for constant monitoring of dynamic cell and tissue culture and growth.
Assuntos
Células-Tronco Pluripotentes Induzidas , Terapia por Ultrassom , Cálcio , Humanos , Miócitos Cardíacos , UltrassonografiaRESUMO
Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 and 633 nm to image lipodamine dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence, respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm2 at both 800 kHz and 1 MHz. Additionally, 800 kHz at 1 W/cm2 was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.
Assuntos
Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos da radiação , Ondas Ultrassônicas , Animais , Feminino , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G(alphai3)-PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas and the G(alphao)-mediated AQP-6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH(+)-ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer-scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH(+)-ATPase in GTP-G(alphao)-mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP-G(alphao)-mediated vesicle swelling is vH(+)-ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin-sensitive vesicle acidification, following the GTP-G(alphao)-induced swelling stimulus. Water channels are bidirectional and the vH(+)-ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggesting that vH(+)-ATPase is upstream of AQP-6, in the pathway leading from G(alphao)-stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6-mediated gating of water into synaptic vesicles.
Assuntos
Adenosina Trifosfatases/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Aquaporina 6/metabolismo , Western Blotting , Exocitose , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Microscopia Eletrônica de Transmissão , Ratos , Ratos Sprague-Dawley , Sinaptossomos/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150 nm in diameter in acinar cells of the exocrine pancreas to 12 nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.
Assuntos
Membrana Celular/metabolismo , Exocitose , Lipoproteínas/metabolismo , Vesículas Secretórias/metabolismo , Animais , Humanos , Modelos Biológicos , Neurônios/metabolismo , Pâncreas Exócrino/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
In the recent years, the Electrocardiogram (ECG) based biometric identification has been a subject of considerable research interest. In this paper, we present non-fiducial method for ECG-identification using the short time Fourier transform (STFT), and Frechet mean distance-based algorithms to find the similarity between the STFTs of different people. In this study, we select randomly the training and test data of the ECG in order to test the stability of the method. We apply our proposed method on 124 ECG records of 62 subjects from the publicly available ECG ID database from physionet website. Our preliminary results indicate that the Frechet mean based ECG identification has 96.45% average identification accuracy and therefore can be potentially useful in various applications.
Assuntos
Identificação Biométrica , Processamento de Sinais Assistido por Computador , Algoritmos , Eletrocardiografia , Análise de FourierRESUMO
The tolerability and efficacy of low-frequency, low-intensity therapeutic ultrasound-induced insulin release was investigated in a pre-clinical in vivo murine model. The treatment groups received a single 5-min continuous sonication at 1 MHz and 1.0 W/cm2. Insulin and glucagon levels in the serum were determined using enzyme-linked immunosorbent assay. The pancreas was excised and sectioned for histologic analysis. In terminal studies, we observed a moderate (â¼50 pM) but significant increase in blood insulin concentration in vivo immediately after sonication compared with a decrease of approximately 60 pM in sham animals (n < 6, p < 0.005). No difference was observed in the change in glucose or glucagon concentrations between groups. Comparisons of hematoxylin and eosin-stained terminal and survival pancreatic tissue revealed no visible differences or evidence of damage. This study is the first step in assessing the translational potential of therapeutic ultrasound as a treatment for early stages of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/terapia , Glucagon/sangue , Insulina/sangue , Terapia por Ultrassom , Animais , Camundongos , Pâncreas/metabolismo , Pâncreas/efeitos da radiação , Distribuição AleatóriaRESUMO
Human Islet Amyloid Polypeptide or amylin is a neuroendocrine peptide with important endocrine and paracrine functions. Excessive production and accumulation of human amylin in the pancreas can lead to its aggregation and apoptosis of islet ß-cells. Amylin has been shown to function within the central nervous system to decrease food intake, and more recently, it has been revealed that amylin is directly transcribed from neurons of the central nervous system, including the hypothalamus, arcuate nucleus, medial preoptic area, and nucleus accumbens. These findings alter the current model of how amylin targets the nervous system, and as a result may lead to obesity and type II diabetes mellitus. Here we set out to use Caenorhabditis elegans as an inducible in vivo model system to study the effects of amylin overexpression in tissues that include the nervous system. We profiled the transcriptional changes in transgenic animals expressing human amylin through RNA-seq. Using this genome-wide approach our results revealed for the first time that expression of human amylin in tissues including the nervous system induce diverse physiological responses in various signaling pathways. From our characterization of transgenic C. elegans animals expressing human amylin, we also observed specific defects in neural developmental programs as well as sensory behavior. Taken together, our data demonstrate the utility of using C. elegans as a valuable in vivo model to study human amylin toxicity.
Assuntos
Caenorhabditis elegans/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Animais , Animais , Animais Geneticamente Modificados , Comportamento Animal , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Locomoção , Neurônios/fisiologia , Transdução de Sinais , Transcrição GênicaRESUMO
Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet ß-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to ß-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic ß-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. ß-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in ß-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic ß-cells.