Abram Katz

Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction‐mediated glucose transport. Repeated contractions increased 2‐deoxyglucose (2‐DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast‐twitch) muscle. N‐Acetylcysteine (NAC), a non‐specific antioxidant, inhibited contraction‐mediated 2‐DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2‐DG uptake or the uptake induced by insulin, hypoxia or 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR, which mimics AMP‐mediated activation of AMP‐activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction‐mediated 2‐DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+‐dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction‐mediated 2‐DG uptake versus muscles from wild‐type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction‐mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10‐fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction‐mediated activation of glucose transport in fast‐twitch muscle.

Skeletal muscle: energy metabolism, fiber types, fatigue and adaptability.

Skeletal muscles cope with a large range of activities, from being able to support the body weight during long periods of upright standing to perform explosive movements in response to an unexpected threat. This requires systems for energy metabolism that can provide energy during long periods of moderately increased energy consumption as well as being able to rapidly increasing the rate of energy production more than 100-fold in response to explosive contractions. In this short review we discuss how muscles can deal with these divergent demands. We first outline the major energy metabolism pathways in skeletal muscle. Next we describe metabolic differences between different muscle fiber types. Contractile performance declines during intense activation, i.e. fatigue develops, and we discuss likely underlying mechanisms. Finally, we discuss the ability of muscle fibers to adapt to altered demands, and mechanisms behind these adaptations. The accumulated experimental evidence forces us to conclude that most aspects of energy metabolism involve multiple and overlapping signaling pathways, which indicates that the control of energy metabolism is too important to depend on one single molecule or mechanism.

Hypermetabolism in mice caused by the central action of an unliganded thyroid hormone receptor α1

Thyroid hormone, via its nuclear receptors TRα and TRβ, controls metabolism by acting locally in peripheral tissues and centrally by regulating sympathetic signaling. We have defined aporeceptor regulation of metabolism by using mice heterozygous for a mutant TRα1 with low affinity to T3. The animals were hypermetabolic, showing strongly reduced fat depots, hyperphagia and resistance to diet‐induced obesity accompanied by induction of genes involved in glucose handling and fatty acid metabolism in liver and adipose tissues. Increased lipid mobilization and β‐oxidation occurred in adipose tissues, whereas blockade of sympathetic signaling to brown adipose tissue normalized the metabolic phenotype despite a continued perturbed hormone signaling in this cell type. The results define a novel and important role for the TRα1 aporeceptor in governing metabolic homeostasis. Furthermore, the data demonstrate that a nuclear hormone receptor affecting sympathetic signaling can override its autonomous effects in peripheral tissues.

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK‐deficient mice (CK_/_). With high‐intensity electrical stimulation, single fibers from CK_/_ mice displayed a transient decrease in both tetanic free myoplasmic [Ca2+] ([Ca2+]i, measured with the fluorescent dye indo‐1) and force that was not observed in wild‐type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast‐twitch, fatigued more slowly in CK_/_ than in wild‐type mice; on the other hand, the slow‐twitch soleus muscle fatigued more rapidly in CK_/_ mice. In single wild‐type fibers, tetanic force decreased and [Ca2+]i increased during the first 10 fatiguing tetani, but this was not observed in CK_/_ fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK_/_ muscles. In conclusion, CK is important for avoiding fatigue at the onset of high‐intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.—Dahlstedt, A. J., Katz, A., Wier‐inga, B., Westerblad, H. Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase. FASEB J. 14, 982–990 (2000)

Effects of Palmitate on Ca2+ 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 Ca2+ 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 Ca2+ transients (measured with fluo-3), the sarcoplasmic reticulum Ca2+ load, and cell shortening by ∼20% in wild-type cardiomyocytes; these decreases were prevented by the general antioxidant N-acetylcysteine. In contrast, palmitate accelerated Ca2+ 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 Ca2+ handling and contraction in a ROS-dependent manner in normal cardiomyocytes. Conversely, high fatty acid levels may be vital to sustain cardiac Ca2+ handling and contraction in obesity and insulin-resistant conditions.

Role of myoplasmic phosphate in contractile function of skeletal muscle: studies on creatine kinase‐deficient mice

Increased myoplasmic inorganic phosphate (Pi) has been suggested to have an important role in skeletal muscle fatigue, especially in the early phase. In the present study we used intact fast‐twitch muscle cells from mice completely deficient in creatine kinase (CK‐/‐) to test this suggestion. These CK‐/‐ muscle cells provide a good model since they display a higher Pi concentration in the unfatigued state and fatigue without significant increase of Pi. Tetanic contractions (350 ms duration) were produced in intact single muscle fibres. The free myoplasmic [Ca2+] ([Ca2+]i) was measured with the fluorescent indicator indo‐1. The force‐[Ca2+]i relationship was constructed from tetani at different frequencies. Compared with wild‐type fibres, CK‐/‐ fibres displayed lower force in 100 Hz tetani and at saturating [Ca2+]i (i.e. 100 Hz stimulation during caffeine exposure), higher tetanic [Ca2+]i during the first 100 ms of tetanic stimulation, reduced myofibrillar Ca2+ sensitivity when measurements were performed 100–200 ms into tetani, and slowed force relaxation that was due to altered cross‐bridge kinetics rather than delayed Ca2+ removal from the myoplasm. In wild‐type fibres, a series of 10 tetani resulted in reduced tetanic force, slowed force relaxation, and increased amplitude of [Ca2+]i tails after tetani. None of these changes were observed in CK‐/‐ fibres. Complementary experiments on isolated fast‐twitch extensor digitorum longus muscles showed a reduction of tetanic force and relaxation speed in CK‐/‐ muscles similar to those observed in single fibres. In conclusion, increased Pi concentration can explain changes observed in the early phase of skeletal muscle fatigue. Increased Pi appears to be involved in both fatigue‐induced changes of cross‐bridge function and SR Ca2+ handling.

Reactive oxygen species and fatigue-induced prolonged low-frequency force depression in skeletal muscle fibres of rats, mice and SOD2 overexpressing mice

Skeletal muscle often shows a delayed force recovery after fatiguing stimulation, especially at low stimulation frequencies. In this study we focus on the role of reactive oxygen species (ROS) in this fatigue‐induced prolonged low‐frequency force depression. Intact, single muscle fibres were dissected from flexor digitorum brevis (FDB) muscles of rats and wild‐type and superoxide dismutase 2 (SOD2) overexpressing mice. Force and myoplasmic free [Ca2+] ([Ca2+]i) were measured. Fibres were stimulated at different frequencies before and 30 min after fatigue induced by repeated tetani. The results show a marked force decrease at low stimulation frequencies 30 min after fatiguing stimulation in all fibres. This decrease was associated with reduced tetanic [Ca2+]i in wild‐type mouse fibres, whereas rat fibres and mouse SOD2 overexpressing fibres instead displayed a decreased myofibrillar Ca2+ sensitivity. The SOD activity was ∼50% lower in wild‐type mouse than in rat FDB muscles. Myoplasmic ROS increased during repeated tetanic stimulation in rat fibres but not in wild‐type mouse fibres. The decreased Ca2+ sensitivity in rat fibres could be partially reversed by application of the reducing agent dithiothreitol, whereas the decrease in tetanic [Ca2+]i in wild‐type mouse fibres was not affected by dithiothreitol or the antioxidant N‐acetylcysteine. In conclusion, we describe two different causes of fatigue‐induced prolonged low‐frequency force depression, which correlate to differences in SOD activity and ROS metabolism. These findings may have clinical implications since ROS‐mediated impairments in myofibrillar function can be counteracted by reductants and antioxidants, whereas changes in SR Ca2+ handling appear more resistant to interventions.

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 [Ca2+] ([Ca2+]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 ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca2+]i, compared with an increase of ∼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 Ca2+ release from the sarcoplasmic reticulum.

Effect of fructose ingestion on muscle glycogen usage during exercise.

Eight healthy males were studied to compare the effects of preexercise fructose and glucose ingestion on muscle glycogen usage during exercise. Subjects performed three randomly assigned trials, each involving 30 min of cycling exercise at 75% VO2max. Forty-five min prior to commencing each trial, subjects ingested either 50 g of glucose (G), 50 g of fructose (F), or sweet placebo (C). No differences in VO2 or respiratory exchange ratio were observed between the trials. Blood glucose was elevated (P less than 0.05) as a result of the glucose feeding. With the onset of exercise, blood glucose declined rapidly during G, reaching a nadir of 3.18 +/- 0.15 (SE) mmol X 1(-1) at 20 min of exercise. This value was lower (P less than 0.05) than the corresponding values in F (3.79 +/- 0.20) and C (3.99 +/- 0.18). No differences in exercise blood glucose levels were observed between F and C. Muscle glycogen utilization was greater (P less than 0.05) during G (55.4 +/- 3.3 mmol X kg-1 w.w.) than C (42.8 +/- 4.2). No difference was observed between F (45.6 +/- 4.3) and C. There was a trend (P = 0.07) for muscle glycogen usage to be lower during F than G. These results suggest that the adverse effects of preexercise glucose ingestion are, in general, not observed with either fructose or sweet placebo.

Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomycytes

Non‐technical summary  When under stress, the heart beat becomes stronger, in part due to enhanced fluxes of Ca2+ at the level of the cardiac cell. It is known that this effect is mediated by activation of β‐receptors on the cardiac cell surface. This leads to modifications of intracellular proteins that in turn increase the flux of Ca2+ within the cell. In this study we show that activation of β‐receptors increases the production of reactive oxygen species (ROS) in the heart cell. These ROS generate enhanced Ca2+ fluxes and more vigorous contraction. This finding shows a new cellular signalling route for regulating the power of the heart beat and might contribute to our understanding of diseases with defective cardiac contraction, such as heart failure.