Anders Arner

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.

Positive Inotropic Effects by Uridine Triphosphate (UTP) and Uridine Diphosphate (UDP) via P2Y2 and P2Y6 Receptors on Cardiomyocytes and Release of UTP in Man During Myocardial Infarction

The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y2/4 agonist UTPγS increased contraction by 52%, similar to β1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPβS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDPβS and the UTPγS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.

Effects of calcium and substrate on force‐velocity relation and energy turnover in skinned smooth muscle of the guinea‐pig.

Mechanical properties and rate of ATP breakdown (JATP) have been determined in the chemically skinned guinea‐pig taenia coli at 22 degrees C. The influence of varied [Ca2+], [Mg ATP] and muscle length were investigated. The shortening response after a step decrease in force (isotonic quick release) was highly curvilinear in the first 100‐200 ms. This effect was shown to be a time‐dependent response to the force step and not primarily caused by the shift along the length‐force relation associated with shortening. Maximal shortening velocity (Vmax) decreased gradually following the release. At pCa (= ‐log [Ca2+]) 4.5, Vmax at 20 and 1000 ms after release was 0.49 +/‐ 0.07 and 0.041 +/‐ 0.004 (mean +/‐ S.E. of mean, n = 5) lengths s‐1 respectively. Unloaded shortening velocity obtained from length steps of different magnitude (slack test) also showed a gradual decrease after the release, consistent with the isotonic release results. Increasing [Ca2+] from the relaxed state at pCa 9 (1 microM‐calmodulin present) gave increased isometric force to a maximum at pCa 4.5. Half‐maximal response was obtained at pCa 6.1. JATP at maximal force at pCa 4.5 was about 3 times the basal rate at pCa 9. The relation between JATP and force was highly non‐linear, with a marked increase in JATP with little alteration in force at the highest [Ca2+]. When force was reduced to zero at pCa 4.5 by shortening the muscle to 0.3 L0 (L0 being the length giving maximal active force), JATP decreased by about 30%. At two levels of [Ca2+] giving similar force (pCa 5.75 and 4.5) the energetic tension cost obtained by length variations was lower at the low [Ca2+]. At pCa 6.0, Vmax and force were decreased to the same extent relative to their values at pCa 4.5. At pCa 5.75, where there was no reduction in force but a 25% decrease in isometric JATP, Vmax was unchanged relative to pCa 4.5. Force, Vmax and JATP were all dependent on [Mg ATP]. Half‐maximal response was obtained at 0.1 mM for force and Vmax, and at 0.5 mM for JATP. The results are discussed in relation to a possible influence of both Ca2+ and Mg ATP on kinetic properties of the cross‐bridge cycle.

Recapitulation of developmental cardiogenesis governs the morphological and functional regeneration of adult newt hearts following injury

Urodele amphibians, like the newt, are the champions of regeneration as they are able to regenerate many body parts and tissues. Previous experiments, however, have suggested that the newt heart has only a limited regeneration capacity, similar to the human heart. Using a novel, reproducible ventricular resection model, we show for the first time that adult newt hearts can fully regenerate without any evidence of scarring. This process is governed by increased proliferation and the up-regulation of cardiac transcription factors normally expressed during developmental cardiogenesis. Furthermore, we are able to identify cells within the newly regenerated regions of the myocardium that express the LIM-homeodomain protein Islet1 and GATA4, transcription factors found in cardiac progenitors. Information acquired from using the newt as a model organism may help to shed light on the regeneration deficits demonstrated in damaged human hearts.

Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions

Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormones central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

Nonmuscle Myosin motor of smooth muscle.

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371–375). Intracellular calcium was measured in urinary bladders from SM-MHC–deficient and SM-MHC–expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC–deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC–deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC–deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC–expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.

Inappropriate heat dissipation ignites brown fat thermogenesis in mice with a mutant thyroid hormone receptor α1

Significance Patients with thyroid disease often report sensitivity to environmental temperature, feeling too hot or too cold in hyper- and hypothyroidism, respectively. Textbook knowledge attributes this to the role of thyroid hormone in basal metabolism. In this study, we show that a mouse model for hypothyroidism lacks proper control of tail vasoconstriction at room temperature, which causes inappropriate heat loss. This in turn activates heat-generating brown adipose tissue to maintain body temperature. Our study therefore demonstrates that changes in vascular control may in fact significantly contribute to the temperature hypersensitivity observed in patients with thyroid disorders and reveal a unique connection between vascular and metabolic effects of thyroid hormone. Thyroid hormone is a major regulator of thermogenesis, acting both in peripheral organs and on central autonomic pathways. Mice heterozygous for a point mutation in thyroid hormone receptor α1 display increased thermogenesis as a consequence of high sympathetic brown fat stimulation. Surprisingly, despite the hypermetabolism, their body temperature is not elevated. Here we show, using isolated tail arteries, that defective thyroid hormone receptor α1 signaling impairs acetylcholine-mediated vascular relaxation as well as phenylephrine-induced vasoconstriction. Using infrared thermography on conscious animals, we demonstrate that these defects severely interfere with appropriate peripheral heat conservation and dissipation, which in turn leads to compensatory alterations in brown fat activity. Consequently, when the vasoconstrictive defect in mice heterozygous for a point mutation in thyroid hormone receptor α1 was reversed with the selective α1-adrenergic agonist midodrine, the inappropriate heat loss over their tail surface was reduced, normalizing brown fat activity and energy expenditure. Our analyses demonstrate that thyroid hormone plays a key role in vascular heat conservation and dissipation processes, adding a unique aspect to its well-documented functions in thermoregulation. The data thus facilitate understanding of temperature hypersensitivity in patients with thyroid disorders. Moreover, the previously unrecognized connection between cardiovascular regulation and metabolic activity revealed in this study challenges the interpretation of several experimental paradigms and questions some of the currently derived hypotheses on the role of thyroid hormone in thermogenesis.

Separate and overlapping metabolic functions of LXRα and LXRβ in C57Bl/6 female mice

The two liver X receptors (LXRs), LXRalpha and LXRbeta, are transcriptional regulators of cholesterol, lipid, and glucose metabolism and are both activated by oxysterols. Impaired metabolism is linked with obesity, insulin resistance, and type 2-diabetes (T2D). In the present study, we aimed to delineate the specific roles of LXRalpha and -beta in metabolic processes. C57Bl/6 female mice were fed a normal or a high-fat diet (HFD) and metabolic responses in wild-type, LXRalpha(-/-), LXRbeta(-/-), and LXRalphabeta(-/-) mice were analyzed. Whole body fat and intramyocellular lipid contents were measured by nuclear magnetic resonance. Energy expenditure was measured in individual metabolic cages. Glucose, insulin, and pyruvate tolerance tests were performed and gene expression profiles analyzed by qPCR. We found that both LXRbeta(-/-) and LXRalphabeta(-/-) mice are resistant to HFD-induced obesity independently of the presence of high cholesterol. Using tolerance tests, we found that, on an HFD, LXRbeta(-/-) mice enhanced their endogenous glucose production and became highly insulin resistant, whereas LXRalpha(-/-) and LXRalphabeta(-/-) mice remained glucose tolerant and insulin sensitive. Gene expression profiling confirmed that LXRbeta is the regulator of lipogenic genes in visceral white adipose tissue (WAT) and muscle tissue and, surprisingly, that Ucp1 and Dio2 are not responsible for the protection against diet-induced obesity observed in LXRbeta(-/-) and LXRalphabeta(-/-) mice. LXRalpha is required for the control of cholesterol metabolism in the liver, while LXRbeta appears to be a major regulator of glucose homeostasis and energy utilization and of fat storage in muscle and WAT. We conclude that selective LXRbeta agonists would be novel pharmaceuticals in the treatment of T2D.

Force-velocity characteristics and active tension in relation to content and orientation of smooth muscle cells in aortas from normotensive and spontaneous hypertensive rats.

Segments of abdominal aorta from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats (20–25 weeks) were compared with respect to force production and dynamic mechanical properties. The preparations were mounted in vitro for determination of optimal length (lo) for active force, and then maximally stimulated (high-K+ solution, 10 mm Ca2+, 10−5 M noradrenaline), and fixed for electron microscopy. Muscle cellular volume per mm vessel wall was significantly (P < 0.01) higher in SHR (0.14 ± 0.009 mm3, n = 7) compared to WKY (0.11 ± 0.004 mm3, n = 7). Unchanged cell length and unaltered cross-sectional area (17μm2) of nucleus containing cell profiles in SHR suggest an increased number of cells in the media. No difference was found in maximum force per unit cell area between SHR (271 ± 31, n = 7) and WKY (305 ± 49 mN/ mm2, n = 7). Cell orientation was almost circular in both groups, showing that force was measured in parallel to the cell long axis. Aortic segments were mounted in an apparatus for quick-release experiments. They were maximally stimulated and force steps were imposed at peak of contractions. The series elastic component, characterized by the initial elastic recoils at 0.75 lo, had similar stiffness values in SHR and WKY. Velocities were measured 100 msec after release. The results were fitted to Hills equation and maximum shortening velocity (Vmax) computed. No difference in Vmax was found at 0.75 lo (WKY: 0.048 ± 0.005; SHR: 0.042 ± 0.006 lo/s, n = 6 for both). At 0.85 lo, the data were corrected for passive tension (40% of total). Vmax at 0.85 l0 was 0.071 ± 0.009 lo/s (n = 5) for WKY, and 0.069 ± 0.007 lo/s (n = 5) for SHR. Similar Vmax and force per cell cross-sectional area suggest similar characteristics of actomyosin interaction in SHR and WKY aorta.

Structure and Function of Skeletal Muscle in Zebrafish Early Larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5–7 d). Using aluminum clips, preparations (∼1.5 mm length, 150 μm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length–force relations showed an optimal sarcomere length of 2.15 μm. x-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an ∼25° angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 × 10−3 μm3, which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (∼186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s−1. In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.