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Joseph D Bruton

Publications and source records attributed to Joseph D Bruton.

10 recordsLinked to original sources
PubMed2007

Effects of palmitate on Ca(2+) handling in adult control and ob/ob cardiomyocytes: impact of mitochondrial reactive oxygen species.

Obesity and insulin resistance are associated with enhanced fatty acid utilization, which may play a central role in diabetic cardiomyopathy. We now assess the effect of the saturated fatty acid palmitate (1.2 mmol/l) on Ca(2+) handling, cell shortening, and mitochondrial production of reactive oxygen species (ROS) in freshly isolated ventricular cardiomyocytes from normal (wild-type) and obese, insulin-resistant ob/ob mice. Cardiomyocytes were electrically stimulated at 1 Hz, and the signal of fluorescent indicators was measured with confocal microscopy. Palmitate decreased the amplitude of cytosolic Ca(2+) transients (measured with fluo-3), the sarcoplasmic reticulum Ca(2+) load, and cell shortening by approximately 20% in wild-type cardiomyocytes; these decreases were prevented by the general antioxidant N-acetylcysteine. In contrast, palmitate accelerated Ca(2+) transients and increased cell shortening in ob/ob cardiomyocytes. Application of palmitate rapidly dissipated the mitochondrial membrane potential (measured with tetra-methyl rhodamine-ethyl ester) and increased the mitochondrial ROS production (measured with MitoSOX Red) in wild-type but not in ob/ob cardiomyocytes. In conclusion, increased saturated fatty acid levels impair cellular Ca(2+) handling and contraction in a ROS-dependent manner in normal cardiomyocytes. Conversely, high fatty acid levels may be vital to sustain cardiac Ca(2+) handling and contraction in obesity and insulin-resistant conditions.

Animals↗
PubMed2006

Insulin potentiates TRPC3-mediated cation currents in normal but not in insulin-resistant mouse cardiomyocytes.

OBJECTIVE: Recent studies show that bioactive lipids alter intracellular Ca(2+) handling of cardiac cells differently in normal and insulin-resistant cardiomyocytes. In the present study we measured non-selective cation currents (NSCC) focusing on the interaction between insulin, the bioactive lipid diacylglycerol (DAG) and canonical transient receptor potential 3 (TRPC3) channels. METHODS: Whole cell patch-clamp was used to measure NSCC in ventricular cardiomyocytes isolated from adult wild-type (WT) and insulin resistant, obese ob/ob mice. Western blot, immunoprecipitation and immunofluorescence staining were used to study the concentration and cellular distribution of TRPC3 channels. RESULTS: Application of the membrane permeable DAG analogue (OAG, 30 microM) induced an NSCC, which was approximately 40% smaller in ob/ob than in WT cardiomyocytes. Insulin induced a small NSCC with similar amplitude in ob/ob and WT cells. Pretreatment with insulin (60 nM) increased the OAG-induced NSCC in WT (by approximately 50%) but not in ob/ob cells. OAG-induced currents were inhibited by adding anti-TRPC3 antibodies to the patch pipette solution. The expression of TRPC3 was lower in ob/ob than in WT cardiomyocytes. TRPC3 was detected in glucose transporter 4 (GLUT4) immunoprecipitates. Insulin exposure resulted in a translocation of TRPC3 to the plasma membrane in WT but not in ob/ob cardiomyocytes. CONCLUSIONS: Insulin-resistant ob/ob cardiomyocytes showed decreases in DAG-mediated NSCC, which were accompanied by decreased TRPC3 expression and defective insulin-mediated trafficking of this protein.

Animals↗
PubMed2006

Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle.

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 mm lactic acid nor increasing the [K(+)] of the bath solution from 5 to 10 mm had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 mm[K(+)]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca(2+)] ([Ca(2+)](i)). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by approximately 80%. The force decrease in the presence of cyanide was associated with a approximately 50% decrease in tetanic [Ca(2+)](i), compared with an increase of approximately 20% without cyanide. In conclusion, lactic acid or [K(+)] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca(2+) release from the sarcoplasmic reticulum.

Action Potentials↗
PubMed2006

The role of Ca2+ influx for insulin-mediated glucose uptake in skeletal muscle.

The involvement of Ca(2+) in insulin-mediated glucose uptake is uncertain. We measured Ca(2+) influx (as Mn(2+) quenching or Ba(2+) influx) and 2-deoxyglucose (2-DG) uptake in single muscle fibers isolated from limbs of adult mice; 2-DG uptake was also measured in isolated whole muscles. Exposure to insulin increased the Ca(2+) influx in single muscle cells. Ca(2+) influx in the presence of insulin was decreased by 2-aminoethoxydiphenyl borate (2-APB) and increased by the membrane-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), agents frequently used to block and activate, respectively, nonselective cation channels. Maneuvers that decreased Ca(2+) influx in the presence of insulin also decreased 2-DG uptake, whereas increased Ca(2+) influx was associated with increased insulin-mediated glucose uptake in isolated single cells and whole muscles from both normal and insulin-resistant obese ob/ob mice. 2-APB and OAG affected neither basal nor hypoxia- or contraction-mediated 2-DG uptake. 2-APB did not inhibit the insulin-mediated activation of protein kinase B or extracellular signal-related kinase 1/2 in whole muscles. In conclusion, alterations in Ca(2+) influx specifically modulate insulin-mediated glucose uptake in both normal and insulin-resistant skeletal muscle. Moreover, the present results indicate that Ca(2+) acts late in the insulin signaling pathway, for instance, in the GLUT4 translocation to the plasma membrane.

Animals↗
PubMed2005

Insulin and inositol 1,4,5-trisphosphate trigger abnormal cytosolic Ca2+ transients and reveal mitochondrial Ca2+ handling defects in cardiomyocytes of ob/ob mice.

Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by approximately 30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.

Animals↗
PubMed2003

Mitochondrial function in intact skeletal muscle fibres of creatine kinase deficient mice.

Creatine kinase (CK) has a central role in skeletal muscle, acting as a fast energy buffer and shuttle between sites of energy production (mitochondria) and consumption (cross-bridges and ion pumps). Unexpectedly, isolated fast-twitch skeletal muscle cells of mice deficient in both cytosolic and mitochondrial CK (CK-/-) are highly fatigue resistant during stimulation protocols that stress aerobic metabolism. We have now studied different aspects of mitochondrial function in CK-/- skeletal muscle. Intact, single fibres of flexor digitorum brevis (FDB) muscles were fatigued by repeated tetanic stimulation (70 Hz, 350 ms duration, duty cycle 0.14). Under control conditions, CK-/- FDB fibres were more fatigue resistant than wild-type fibres. However, after mitochondrial inhibition with cyanide, force declined markedly faster in CK-/- fibres than in wild-type fibres. The rapid force decline in CK-/- fibres was not due to decreased myoplasmic [Ca2+] during tetani (measured with indo-1), which in these fibres remained virtually constant during fatigue in the presence of cyanide. Intact, single fibres of highly oxidative soleus muscles were fatigued by repeated tetani (50 Hz, 500 ms duration, duty cycle 0.5). All CK-/- soleus fibres tested (n = 9) produced > 40 % force at the end of the fatiguing stimulation period (500 tetani), whereas force fell to < 40 % before 500 tetani in two of three wild-type fibres. Mitochondrial [Ca2+] (measured with rhod-2 and confocal microscopy) increased during repeated tetanic stimulation in CK-/- but not in wild-type FDB fibres. In conclusion, mitochondria and energy shuttling operate effectively in CK-/- fibres and this is associated with an increase in mitochondrial [Ca2+].

Animals↗
PubMed2002

Ryanodine receptors of pancreatic beta-cells mediate a distinct context-dependent signal for insulin secretion.

The ryanodine (RY) receptors in beta-cells amplify signals by Ca2+-induced Ca2+ release (CICR). The role of CICR in insulin secretion remains unclear in spite of the fact that caffeine is known to stimulate secretion. This effect of caffeine is attributed solely to the inhibition of cAMP-phosphodiesterases (cAMP-PDEs). We demonstrate that stimulation of insulin secretion by caffeine is due to a sensitization of the RY receptors. The dose-response relationship of caffeine-induced inhibition of cAMP-PDEs was not correlated with the stimulation of insulin secretion. Sensitization of the RY receptors stimulated insulin secretion in a context-dependent manner, that is, only in the presence of a high concentration of glucose. This effect of caffeine depended on an increase in [Ca2+]i. Confocal images of beta-cells demonstrated an increase in [Ca2+]i induced by caffeine but not by forskolin. 9-Methyl-7-bromoeudistomin D (MBED), which sensitizes RY receptors, did not inhibit cAMP-PDEs, but it stimulated secretion in a glucose-dependent manner. The stimulation of secretion by caffeine and MBED involved both the first and the second phases of secretion. We conclude that the RY receptors of beta-cells mediate a distinct glucose-dependent signal for insulin secretion and may be a target for developing drugs that will stimulate insulin secretion only in a glucose-dependent manner.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Regulation of myoplasmic Ca(2+) in genetically obese (ob/ob) mouse single skeletal muscle fibres.

The present study examined whether calcium handling in skeletal muscle fibres from ob/ob mice was abnormal compared to normal mice. Simultaneous measurements of free myoplasmic calcium and force were made in mouse single intact muscle fibres at rest, during repetitive stimulation and for 30 min afterwards. Fibres were subjected to two bouts of intermittent tetanic contractions 1 h apart. The first bout consisted of 50 tetani only, while during the second bout stimulation was continued until force fell to 40% of control. During a bout of 50 repeated contractions, muscle fibres from ob/ob mice were unable to maintain basal calcium and tetanic calcium transients. During a second series of contractions, muscle fibres from ob/ob mice showed a marked improvement in calcium handling compared to the first series but still fatigued more rapidly than control fibres. It is concluded that calcium handling in skeletal muscle fibres from ob/ob mice is abnormal compared to fibres from normal mice and this contributes to premature fatigue.

Animals↗
PubMed2002

Dynamic vacuolation in skeletal muscle fibres after fatigue.

Vacuoles develop after fatiguing stimulation in frog skeletal muscle fibres. Experiments on isolated Xenopus muscle fibres show that this vacuolation is a dynamic process that reaches its maximum about 20 min after the end of fatiguing stimulation and then recedes. Fatigue-induced vacuoles originate from the t-tubular system. Recent data indicate that vacuoles are formed because of lactate accumulation in the t-tubules resulting in increased osmotic pressure and subsequent water influx. There is no obligatory connection between the presence of vacuoles and force depression, which is another common feature during the recovery from fatigue. Nevertheless, extensive vacuolation may exaggerate this force depression.

Animals↗