RESUMO
AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Glucose/metabolismo , Secreção de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
To improve the imaging performance of optical projection tomography (OPT) in live samples, we have explored a parallelized implementation of semi-confocal line illumination and detection to discriminate against scattered photons. Slice-illuminated OPT (sl-OPT) improves reconstruction quality in scattering samples by reducing interpixel crosstalk at the cost of increased acquisition time. For in vivo imaging, this can be ameliorated through the use of compressed sensing on angularly undersampled OPT data sets. Here, we demonstrate sl-OPT applied to 3D imaging of bead phantoms and live adult zebrafish.
RESUMO
The present study was a 4-week randomized trial to assess the efficacy and safety of sitagliptin, a dipeptidyl-peptidase-4 inhibitor, in persistent or recurring type 2 diabetes after Roux-en-Y gastric bypass surgery (RYGB). Participants (n = 32) completed a mixed meal test (MMT) and self-monitoring of plasma glucose (SMPG) before and 4 weeks after randomization to either sitagliptin 100 mg daily or placebo daily. Questionnaires were administered to assess gastrointestinal discomfort. Outcome variables were glucose, active glucagon-like peptide-1 and ß-cell function during the MMT, and glucose levels during SMPG. Age (56.3 ± 8.2 years), body mass index (34.4 ± 6.7 kg/m2 ), glycated haemoglobin (7.21 ± 0.77%), diabetes duration (12.9 ± 10.0 years), years since RYGB (5.6 ± 3.3 years) and ß-cell function did not differ between the placebo and sitagliptin groups at pre-intervention. Sitagliptin was well tolerated, decreased postprandial glucose levels during the MMT (from 8.31 ± 1.92 mmol/L to 7.67 ± 1.59 mmol/L, P = 0.03) and mean SMPG levels, but had no effect on ß-cell function. In patients with diabetes and mild hyperglycemia after RYGB, a short course of sitagliptin provided a small but significant glucose-lowering effect, with no identified improvement in ß-cell function.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/cirurgia , Derivação Gástrica , Fosfato de Sitagliptina/farmacologia , Adulto , Idoso , Glicemia/metabolismo , Terapia Combinada , Diabetes Mellitus Tipo 2/sangue , Feminino , Hemoglobinas Glicadas/efeitos dos fármacos , Hemoglobinas Glicadas/metabolismo , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fosfato de Sitagliptina/uso terapêuticoRESUMO
Type 2 diabetes (T2D) is characterized by ß cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired ß cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the ß cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ß cells from the earliest expression of the Ins1 gene (â¼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (â¼20%) and Glp1r (â¼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual ß cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed â¼30% decrease in ß cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ß cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Pâncreas/fisiopatologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica/veterinária , Modelos Animais de Doenças , Deleção de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Insulina/sangue , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/patologia , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Pâncreas/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização WntRESUMO
AIMS/HYPOTHESIS: Per-Arnt-Sim kinase (PASK) is a nutrient-regulated domain-containing protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of PASK in the control of islet hormone release, by generating mice with selective deletion of the Pask gene in pancreatic beta or alpha cells. METHODS: Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1 (Cre); PaskBKO) or alpha (Ppg (Cre) [also known as Gcg]; PaskAKO) cell-selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression and islet cell mass were measured using standard techniques. RESULTS: Ins1 (Cre)-based recombination led to efficient beta cell-targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p < 0.05) compared with controls in PaskBKO mice, as well as in global Pask-null mice (38%, p < 0.05). PaskBKO mice displayed normal body weight and fasting glycaemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high-fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycaemic clamps. Although alpha cell mass was increased (21.9%, p < 0.05), glucagon release at low glucose was impaired (p < 0.05) in PaskAKO islets. CONCLUSIONS/INTERPRETATION: The findings demonstrate cell-autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differences between the glycaemic phenotype of global vs cell type-specific null mice suggest important roles for tissue interactions in the control of glycaemia by PASK.
Assuntos
Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Alelos , Animais , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Homeostase/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Fully differentiated pancreatic ß cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess ("glucotoxicity") is implicated in this process, we sought here to identify the potential roles in ß-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK). Highly ß-cell-restricted deletion of each kinase in mice, using an Ins1-controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0-12-fold, E<0.01) increased the expression of subsets of hepatic (Alb, Iyd, Elovl2) and neuronal (Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamate signaling. These changes were partially recapitulated by the loss of AMPK, which also up-regulated ß-cell "disallowed" genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8- to 3.4-fold (E < 0.01). Correspondingly, targeted promoters were enriched for neuronal (Zfp206; P = 1.3 × 10(-33)) and hypoxia-regulated (HIF1; P = 2.5 × 10(-16)) transcription factors. In summary, LKB1 and AMPK, through only partly overlapping mechanisms, maintain ß-cell identity by suppressing alternate pathways leading to neuronal, hepatic, and other characteristics. Selective targeting of these enzymes may provide a new approach to maintaining ß-cell function in some forms of diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
We describe an angular multiplexing technique for optical projection tomography that improves resolution, signal-to-noise ratio, and imaging speed by ameliorating the trade-off between spatial resolution and depth of field and improving the light collection efficiency. Here we demonstrate that imaging at two orthogonal angular projections simultaneously, focused on shifted planes in the sample, improves the average spatial resolution by ~20% and the light collection efficiency by a factor of ~4, thereby enabling increased acquisition speed and reduced light dose.
Assuntos
Tomografia Óptica/métodos , Animais , Processamento de Imagem Assistida por Computador , Cauda , Fatores de Tempo , Peixe-ZebraRESUMO
INTRODUCTION: Single-port laparoscopy (SPL) employs a 1.5- to 2.5-cm incision at the umbilicus for the placement of a single working port. We hypothesized that the longer incision created by SPL compared with multiport laparoscopy may increase the incidence of trocar-site hernias. We examined our experience with SPL in bariatric operations. METHODS: There were 734 laparoscopic sleeve gastrectomy and laparoscopic adjustable gastric banding procedures performed at our institution between 2001 and 2011. Fifty-eight patients were lost to follow-up or had a short duration of follow-up (<1 month). Of the remaining 676 cases, 163 were performed via SPL. All laparoscopic wounds created by trocar size greater than 12 mm were closed with absorbable suture. RESULTS: Patient demographics of the SPL group and the multiport group were similar in terms of age, gender, and comorbidities. The average body mass index (BMI) of the SPL group was lower than the multiport group (43.5 ± 5.3 vs. 45.8 ± 7.7, p < 0.01). The mean follow-up for the SPL group was 11 months versus 24 months for the multiport group. There were three trocar-site hernias out of 513 cases in the multiport compared to one hernia out of 163 cases in the SPL group (0.6 vs. 0.6 %, p = 0.967). All trocar-site hernias occurred at the 15-mm port site. The median time to hernia occurrence for the multiport group was 13 months (range, 1-18). In the SPL group, the hernia occurred at 8 months. On multivariate analysis, age, BMI, SPL, procedure type, and the postoperative weight loss were not associated with the development of trocar-site hernias. CONCLUSIONS: SPL did not increase the rate of trocar-site hernia in this series. A low rate of trocar-site hernia can be achieved with the use of SPL in bariatric surgery.
Assuntos
Cirurgia Bariátrica/métodos , Hérnia Ventral/epidemiologia , Hérnia Ventral/etiologia , Laparoscopia/efeitos adversos , Laparoscopia/métodos , Adulto , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
A single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic biological samples is presented. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio, but the sequential acquisition of projection images typically requires samples to be immobilized during the acquisition. A proof-of-principle system capable of single-shot tomography of a ~1 mm3 volume is presented, demonstrating camera-limited rates of up to 62.5 volumes/s, which has been applied to 3D imaging of a freely swimming zebrafish embryo. This is achieved by recording eight projection views simultaneously on four low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under £5000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including high throughput flow cytometry applied to model organisms and fluid dynamics studies.
Assuntos
Imageamento Tridimensional , Tomografia Óptica , Animais , Imageamento Tridimensional/métodos , Peixe-Zebra , Tomografia Óptica/métodos , Embrião de MamíferosRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.
Assuntos
Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Camundongos , Utrofina/genética , Utrofina/metabolismo , Músculo Esquelético/metabolismo , Sistema de Sinalização das MAP Quinases , Distrofia Muscular de Duchenne/genética , Expressão GênicaRESUMO
We demonstrate two techniques to improve the quality of reconstructed optical projection tomography (OPT) images using the modulation transfer function (MTF) as a function of defocus experimentally determined from tilted knife-edge measurements. The first employs a 2-D binary filter based on the MTF frequency cut-off as an additional filter during back-projection reconstruction that restricts the high frequency information to the region around the focal plane and progressively decreases the spatial frequency bandwidth with defocus. This helps to suppress "streak" artifacts in OPT data acquired at reduced angular sampling, thereby facilitating faster OPT acquisitions. This method is shown to reduce the average background by approximately 72% for an NA of 0.09 and by approximately 38% for an NA of 0.07 compared to standard filtered back-projection. As a biological illustration, a Fli:GFP transgenic zebrafish embryo (3 days post-fertilisation) was imaged to demonstrate the improved imaging speed (a quarter of the acquisition time). The second method uses the MTF to produce an appropriate deconvolution filter that can be used to correct for the spatial frequency modulation applied by the imaging system.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Tomografia Óptica/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.
Assuntos
Epigênese Genética , Impressão Genômica , Animais , Variação Biológica da População , Metilação de DNA , Dieta Hiperlipídica , Feminino , Mamíferos , Camundongos , GravidezRESUMO
A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/análise , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Humanos , Microscopia de Fluorescência , Ligação Proteica , Rodaminas/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/análiseRESUMO
We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisition. Reconstructed image data are presented and compared with results obtained by using the telegraph equation approximation. The new approach provides improved recovery of absorption and scattering parameters while returning similar values for the fluorescence parameters.
Assuntos
Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Óptica e Fotônica/métodos , Animais , Fluorescência , Imageamento Tridimensional/métodos , Camundongos , Modelos Teóricos , Espalhamento de Radiação , Tomografia Óptica/métodosRESUMO
The transcriptional effector SMAD4 is a core component of the TGF-ß family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-ß family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-ß family ligands, which has implications for diseases where Smad4 is mutated or deleted.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Nodal/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Desenvolvimento Embrionário , Endoderma/metabolismo , Técnicas de Inativação de Genes , Mesoderma/metabolismo , Morfogênese , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic beta-cell and hypothalamic neurons using the rat insulin promoter (RIP2).Cre transgene (betaAMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the beta-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the beta-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to betaAMPKdKO mice, betaLKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1-CreER mice, total beta-cell mass and the size of individual islets and beta-cells were increased and islet architecture was markedly altered in betaLKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular Ca(2+) were sharply reduced in betaLKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on beta-cell function.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Western Blotting , Peso Corporal/fisiologia , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Ingestão de Alimentos/fisiologia , Teste de Tolerância a Glucose , Insulina/sangue , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Organismos Livres de Patógenos Específicos , TransgenesRESUMO
We report a three-dimensional time-resolved tomographic imaging technique for localizing protein-protein interaction and protein conformational changes in turbid media based on Förster resonant energy-transfer read out using fluorescence lifetime. This application of "tomoFRET" employs an inverse scattering algorithm utilizing the diffusion approximation to the radiative-transfer equation applied to a large tomographic data set of time-gated images. The approach is demonstrated by imaging a highly scattering cylindrical phantom within which are two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically encoded calcium Förster resonant energy-transfer sensor. A 10 mM calcium chloride solution was added to one of the wells, inducing a protein conformation change upon binding to TN-L15, resulting in Förster resonant energy transfer and a corresponding decrease in the donor fluorescence lifetime. We successfully reconstruct spatially resolved maps of the resulting fluorescence lifetime distribution as well as of the quantum efficiency, absorption, and scattering coefficients.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Óptica e Fotônica/métodos , Cloreto de Cálcio/farmacologia , Linhagem Celular , Citosol/metabolismo , Difusão , Humanos , Imagens de Fantasmas , Conformação Proteica , Espalhamento de Radiação , Silicones/química , Fatores de TempoRESUMO
Of current obesity treatments, bariatric surgery induces the most weight loss. Given the marked increase in the number of bariatric surgeries performed, elucidating the mechanisms of action is a key research goal. We compared whole brain activation in response to high-energy dense (HED) vs. low-energy dense (LED) visual and auditory food cues before and approximately 4â¯months after Roux-en-Y Gastric Bypass (RYGB) (nâ¯=â¯16) and Sleeve Gastrectomy (SG) (nâ¯=â¯9). We included two control groups: a low-calorie diet weight loss group (WL) (nâ¯=â¯14) and a non-treatment group (NT) (nâ¯=â¯16). Relative to the control groups, the surgery groups showed increased dorsolateral prefrontal cortex (dlPFC) and decreased parahippocampal/fusiform gyrus (PHG/fusiform) activation in response to HED vs. LED, suggesting greater cognitive dietary inhibition and decreased rewarding effects and attention related to HED foods. dlPFC activation was significantly more increased in RYGB vs. SG. We also found that postprandial increases in GLP-1 concentrations (pre to postsurgery) correlated with postsurgical decreases in RYGB brain activity in the inferior temporal gyrus and the right middle occipital gyrus in addition to increases in the right medial prefrontal gyrus/paracingulate for HEDâ¯>â¯LED stimuli, suggesting involvement of these attention and inhibitory regions in satiety signaling postsurgery.
Assuntos
Encéfalo/diagnóstico por imagem , Gastrectomia , Derivação Gástrica , Obesidade Mórbida/cirurgia , Estimulação Acústica , Adulto , Feminino , Alimentos , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/diagnóstico por imagem , Estimulação Luminosa , Período Pós-Operatório , Adulto JovemRESUMO
OBJECTIVE: Hepatic insulin clearance is a significant regulator of glucose homestasis. We hypothesized that the improvement in insulin clearance rates (ICRs) under fasting conditions and in response to oral and intravenous (IV) glucose would improve similarly after Roux-en-Y gastric bypass (RYGB) and adjustable gastric banding (AGB) as a function of weight loss; the difference in ICR after oral and IV glucose stimulation will be enhanced after RYGB compared with AGB, an effect mediated by glucagon-like peptide 1 (GLP-1). RESEARCH DESIGN AND METHODS: In study 1, the ICR was calculated under fasting condition (F-ICR), after oral glucose (O-ICR), and after an isoglycemic IV glucose clamp (IV-ICR) in individuals from an established cohort with type 2 diabetes mellitus (T2DM) before, after 10% matched weight loss, and 1 year after either RYGB (n = 22) or AGB (n = 12). In study 2, O-ICR was studied in a separate cohort of individuals with T2DM (n = 22), before and 3 months after RYGB, with and without exendin(9-39) infusion. RESULTS: In study 1, age, BMI, T2DM duration and control, and ICR did not differ between RYGB and AGB preintervention. Weight loss at 1 year was two times greater after RYGB than after AGB (31.6 ± 5.9% vs. 16.6 ± 9.8%; P < 0.05). RYGB and AGB both significantly increased F-ICR, O-ICR, and IV-ICR at 1 year. ICR was inversely associated with insulinemia. The difference between IV-ICR and O-ICR was significantly greater after RYGB versus AGB. GLP-1 antagonism with exendin(9-39) led to an increase in O-ICR in subjects post-RYGB. CONCLUSIONS: Weight loss increased ICR, an effect more pronounced after RYGB compared with AGB. Our data support a potential role for endogenous GLP-1 in the control of postprandial ICR after RYGB.
Assuntos
Diabetes Mellitus Tipo 2/cirurgia , Derivação Gástrica , Gastroplastia , Glucose/administração & dosagem , Insulina/metabolismo , Redução de Peso/fisiologia , Administração Intravenosa , Administração Oral , Adulto , Cirurgia Bariátrica/reabilitação , Estudos de Coortes , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Derivação Gástrica/reabilitação , Gastroplastia/reabilitação , Teste de Tolerância a Glucose , Humanos , Resistência à Insulina/fisiologia , Fígado/metabolismo , Estudos Longitudinais , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Período Pós-PrandialRESUMO
In order to study physical relationships within tissue volumes or even organism-level systems, the spatial distribution of multiple fluorescent markers needs to be resolved efficiently in three dimensions. Here, rather than acquiring discrete spectral images sequentially using multiple emission filters, a hyperspectral scanning laser optical tomography system is developed to obtain hyperspectral volumetric data sets with 2-nm spectral resolution of optically transparent mesoscopic (millimeter-centimeter) specimens. This is achieved by acquiring a series of point-scanning hyperspectral extended depth of field images at different angles and subsequently tomographically reconstructing the 3D intensity distribution for each wavelength. This technique is demonstrated to provide robust measurements via the comparison of spectral and intensity profiles of fluorescent bead phantoms. Due to its enhanced spectral resolving ability, this technique is also demonstrated to resolve largely overlapping fluorophores, as demonstrated by the 3D fluorescence hyperspectral reconstruction of a dual-labeled mouse thymus gland sample and the ability to distinguish tumorous and normal tissues of an unlabeled mouse intestine sample.