Tomáš Špaček - Academia.edu (original) (raw)

Papers by Tomáš Špaček

Research paper thumbnail of Nkx6.1 decline accompanies mitochondrial DNA reduction but subtle nucleoid size decrease in pancreatic islet β-cells of diabetic Goto Kakizaki rats

Scientific reports, Jan 15, 2017

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of... more Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured "nucleoids" which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell ...

Research paper thumbnail of Mitochondrial DNA Nucleoid Distribution at Simulated Pathologies as Visualized by 3D Super-Resolution Biplane FPALM / dSTORM Microscopy

Biophysical Journal, 2014

Research paper thumbnail of Assessment of mitochondrial DNA as an indicator of islet quality: an example in Goto Kakizaki rats

Transplantation proceedings, 2011

Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit a... more Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting β-cells. Among the remaining β-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. β-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn(2+) signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining β-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, r...

Research paper thumbnail of Mitochondrial cristae remodeling in HepG2 cells adapted to hypoxia

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

Research paper thumbnail of Mitochondrial nucleoids as revealed by 3D super-resolution microscopy

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

Research paper thumbnail of Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction

The International Journal of Biochemistry & Cell Biology, 2005

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as in... more Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.

Research paper thumbnail of Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells

The International Journal of Biochemistry & Cell Biology, 2013

Mitochondrial DNA nucleoids 15 Mitochondrial fission 16 Mitochondrial network fragmentation 17 Mi... more Mitochondrial DNA nucleoids 15 Mitochondrial fission 16 Mitochondrial network fragmentation 17 Mitochondrial network reintegration 18 a b s t r a c t

Research paper thumbnail of Oxidative Stress Plays a Major Role in Mitochondrial Nucleoid Clustering

Free Radical Biology and Medicine, 2011

Research paper thumbnail of Mitochondrial Nucleoid Clustering Is Regulated by H2O2 Production in the Respiratory Chain

Free Radical Biology and Medicine, 2012

Research paper thumbnail of Mitochondrial DNA Nucleoid Redistribution after Mitochondrial Network Fragmentation as Visualized by 3D Super-Resolution Biplane Fpalm Microscopy

Biophysical Journal, 2013

Research paper thumbnail of S8.4 Mitochondrial superoxide generation is diminished during glucose-stimulated insulin secretion in INS-1E cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008

Research paper thumbnail of Bi-plane FPALM nanoscopy confirms that mitochondrial nucleoids play a central role in mitochondrial biogenesis

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012

Research paper thumbnail of 4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells, an experimental model of type-2 diabetes

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

3D morphology of mitochondrial network 4Pi microscopy 3D image analysis Pancreatic β-cell Type-2 ... more 3D morphology of mitochondrial network 4Pi microscopy 3D image analysis Pancreatic β-cell Type-2 diabetes Morphological diagnostic Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ∼ 7-fold improved axial resolution (∼ 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redoxsensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.

Research paper thumbnail of Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

The International Journal of Biochemistry & Cell Biology, 2008

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represen... more Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic ␤-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential ( Ψ m ) during GSIS are lacking. Using high resolution respirometry and parallel Ψ m monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and Ψ m during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, Ψ m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20 mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.

Research paper thumbnail of Nkx6.1 decline accompanies mitochondrial DNA reduction but subtle nucleoid size decrease in pancreatic islet β-cells of diabetic Goto Kakizaki rats

Scientific reports, Jan 15, 2017

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of... more Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured "nucleoids" which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell ...

Research paper thumbnail of Mitochondrial DNA Nucleoid Distribution at Simulated Pathologies as Visualized by 3D Super-Resolution Biplane FPALM / dSTORM Microscopy

Biophysical Journal, 2014

Research paper thumbnail of Assessment of mitochondrial DNA as an indicator of islet quality: an example in Goto Kakizaki rats

Transplantation proceedings, 2011

Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit a... more Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting β-cells. Among the remaining β-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. β-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn(2+) signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining β-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, r...

Research paper thumbnail of Mitochondrial cristae remodeling in HepG2 cells adapted to hypoxia

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

Research paper thumbnail of Mitochondrial nucleoids as revealed by 3D super-resolution microscopy

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014

Research paper thumbnail of Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction

The International Journal of Biochemistry & Cell Biology, 2005

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as in... more Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.

Research paper thumbnail of Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells

The International Journal of Biochemistry & Cell Biology, 2013

Mitochondrial DNA nucleoids 15 Mitochondrial fission 16 Mitochondrial network fragmentation 17 Mi... more Mitochondrial DNA nucleoids 15 Mitochondrial fission 16 Mitochondrial network fragmentation 17 Mitochondrial network reintegration 18 a b s t r a c t

Research paper thumbnail of Oxidative Stress Plays a Major Role in Mitochondrial Nucleoid Clustering

Free Radical Biology and Medicine, 2011

Research paper thumbnail of Mitochondrial Nucleoid Clustering Is Regulated by H2O2 Production in the Respiratory Chain

Free Radical Biology and Medicine, 2012

Research paper thumbnail of Mitochondrial DNA Nucleoid Redistribution after Mitochondrial Network Fragmentation as Visualized by 3D Super-Resolution Biplane Fpalm Microscopy

Biophysical Journal, 2013

Research paper thumbnail of S8.4 Mitochondrial superoxide generation is diminished during glucose-stimulated insulin secretion in INS-1E cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008

Research paper thumbnail of Bi-plane FPALM nanoscopy confirms that mitochondrial nucleoids play a central role in mitochondrial biogenesis

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012

Research paper thumbnail of 4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells, an experimental model of type-2 diabetes

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010

3D morphology of mitochondrial network 4Pi microscopy 3D image analysis Pancreatic β-cell Type-2 ... more 3D morphology of mitochondrial network 4Pi microscopy 3D image analysis Pancreatic β-cell Type-2 diabetes Morphological diagnostic Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ∼ 7-fold improved axial resolution (∼ 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redoxsensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.

Research paper thumbnail of Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

The International Journal of Biochemistry & Cell Biology, 2008

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represen... more Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic ␤-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential ( Ψ m ) during GSIS are lacking. Using high resolution respirometry and parallel Ψ m monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and Ψ m during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, Ψ m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20 mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.