Journal of Physiology

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Exercise training decreases mitogen-activated protein kinase phosphatase-3 expression and suppresses hepatic gluconeogenesis in obese mice

15 March 2014
Key points summary

  • When the hepatic insulin signaling is compromised, there is an inadequate suppression of gluconeogenic pathways, leading the organism to high levels of glucose.

  • Studies with animals with obesity induced by high fat diet or genetically modified showed increased MKP-3 expression and MKP-3/Foxo1 association in liver, with a consequent increase in blood glucose concentration, development of insulin resistance and DM2.

  • As a non-pharmacological strategy recognized and indicated for prevention and treatment of diabetes is the regular practice of physical exercise.

  • In this study we demostrated that physical training is an important tool capable of reducing insulin resistance in the liver by reducing the inflammatory process, including the inhibition of MKP-3 and, therefore, suppress gluconeogenic program in obesity rats.

  • The understanding of these new mechanisms by which physical training regulates glucose homeostasis has critical importance to health professionals for the understanding and prevention of diabetes.

  • Abstract

    Insulin plays an important role in the control of hepatic glucose production. Insulin resistant states are commonly associated with excessive hepatic glucose production, which contributes to both fasting hyperglycaemia and exaggerated postprandial hyperglycaemia. In this regard, increased activity of phosphatases may contribute to the dysregulation of gluconeogenesis. Mitogen-activated protein kinase phosphatase-3 (MKP-3) is a key protein involved in the control of gluconeogenesis. MKP-3-mediated dephosphorylation activates FoxO1 (a member of the forkhead family of transcription factors) and subsequently promotes its nuclear translocation and binding to the promoters of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). In this study, we investigated the effects of exercise training on the expression of MKP-3 and its interaction with FoxO1 in the livers of obese animals. We found that exercised obese mice had a lower expression of MKP-3 and FoxO1/MKP-3 association in the liver. Further, the exercise training decreased FoxO1 phosphorylation and protein levels of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and gluconeogenic enzymes (PEPCK and G6Pase). These molecular results were accompanied by physiological changes, including increased insulin sensitivity and reduced hyperglycaemia, which were not caused by reductions in total body mass. Similar results were also observed with oligonucleotide antisense (ASO) treatment. However, our results showed that only exercise training could reduce an obesity-induced increase in HNF-4α protein levels while ASO treatment alone had no effect. These findings could explain, at least in part, why additive effects of exercise training treatment and ASO treatment were not observed. Finally, the suppressive effects of exercise training on MKP-3 protein levels appear to be related, at least in part, to the reduced phosphorylation of Extracellular signal-regulated kinases (ERK) in the livers of obese mice.

    Omega-3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle

    15 March 2014

    Studies have shown increased incorporation of omega-3 fatty acids into whole skeletal muscle following supplementation, although little has been done to investigate the potential impact on the fatty acid composition of mitochondrial membranes and the functional consequences on mitochondrial bioenergetics. Therefore, we supplemented young healthy male subjects (n = 18) with fish oils [2 g eicosapentaenoic acid (EPA) and 1 g docosahexanoic acid (DHA) per day] for 12 weeks and skeletal muscle biopsies were taken prior to (Pre) and following (Post) supplementation for the analysis of mitochondrial membrane phospholipid composition and various assessments of mitochondrial bioenergetics. Total EPA and DHA content in mitochondrial membranes increased (P < 0.05) ~450 and ~320%, respectively, and displaced some omega-6 species in several phospholipid populations. Mitochondrial respiration, determined in permeabilized muscle fibres, demonstrated no change in maximal substrate-supported respiration, or in the sensitivity (apparent Km) and maximal capacity for pyruvate-supported respiration. In contrast, mitochondrial responses during ADP titrations demonstrated an enhanced ADP sensitivity (decreased apparent Km) that was independent of the creatine kinase shuttle. As the content of ANT1, ANT2, and subunits of the electron transport chain were unaltered by supplementation, these data suggest that prolonged omega-3 intake improves ADP kinetics in human skeletal muscle mitochondria through alterations in membrane structure and/or post-translational modification of ATP synthase and ANT isoforms. Omega-3 supplementation also increased the capacity for mitochondrial reactive oxygen species emission without altering the content of oxidative products, suggesting the absence of oxidative damage. The current data strongly emphasize a role for omega-3s in reorganizing the composition of mitochondrial membranes while promoting improvements in ADP sensitivity.

    Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling

    15 March 2014

    Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation–contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (MstnCmpt-dl1Abc or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa–force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca2+ concentration ([Ca2+]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nm, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nm, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 F/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mMs–1) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mm s–1) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

    Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue

    15 March 2014

    The total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu180 causes a lethal form of recessively inherited nemaline myopathy (Amish nemaline myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused an approximately 60% decrease in ssTnT protein expression whereas cre-loxP-mediated deletion of exons 11–13 resulted in total loss of ssTnT, as seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT-deficient soleus muscle also contains significant numbers of small-sized central nuclei type I fibres, indicating active regeneration. The data provide strong support for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved.

    Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle

    15 March 2014

    The Ca2+ uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca2+] heavily buffered in the pCa range (–log10[Ca2+]) 7.3–6.0 for set times and the amount of net SR Ca2+ accumulation determined from the force response elicited upon emptying the SR of all Ca2+. Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca2+ uptake rate at ~pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca2+ uptake rate at ~pCa 6.6. Maximal Ca2+ uptake rate was ~0.18 and ~0.21 mmol Ca2+ (l fibre)–1 s–1 in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg2+] from 1 to 3 mm had no significant effect on the net Ca2+ uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca2+ release channels during uptake in either fibre type. Ca2+ leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ~2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca2+ leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca2+ uptake in the type I human fibres can account for the higher relative level of SR Ca2+ loading observed in type I compared to type II fibres, and the SR Ca2+ leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

    Resting pulmonary haemodynamics and shunting: a comparison of sea-level inhabitants to high altitude Sherpas

    15 March 2014

    The incidence of blood flow through intracardiac shunt and intrapulmonary arteriovenous anastomoses (IPAVA) may differ between Sherpas permanently residing at high altitude (HA) and sea-level (SL) inhabitants as a result of evolutionary pressure to improve gas exchange and/or resting pulmonary haemodynamics. To test this hypothesis we compared sea-level inhabitants at SL (SL-SL; n = 17), during acute isocapnic hypoxia (SL-HX; n = 7) and following 3 weeks at 5050 m (SL-HA; n = 8 non-PFO subjects) to Sherpas at 5050 m (n = 14). S pO 2, heart rate, pulmonary artery systolic pressure (PASP) and cardiac index (Qi) were measured during 5 min of room air breathing at SL and HA, during 20 min of isocapnic hypoxia (SL-HX; P ETO 2 = 47 mmHg) and during 5 min of hyperoxia (F IO 2 = 1.0; Sherpas only). Intracardiac shunt and IPAVA blood flow was evaluated by agitated saline contrast echocardiography. Although PASP was similar between groups at HA (Sherpas: 30.0 ± 6.0 mmHg; SL-HA: 32.7 ± 4.2 mmHg; P = 0.27), it was greater than SL-SL (19.4 ± 2.1 mmHg; P < 0.001). The proportion of subjects with intracardiac shunt was similar between groups (SL-SL: 41%; Sherpas: 50%). In the remaining subjects, IPAVA blood flow was found in 100% of subjects during acute isocapnic hypoxia at SL, but in only 4 of 7 Sherpas and 1 of 8 SL-HA subjects at rest. In conclusion, differences in resting pulmonary vascular regulation, intracardiac shunt and IPAVA blood flow do not appear to account for any adaptation to HA in Sherpas. Despite elevated pulmonary pressures and profound hypoxaemia, IPAVA blood flow in all subjects at HA was lower than expected compared to acute normobaric hypoxia.

    Acute pancreatitis decreases the sensitivity of pancreas-projecting dorsal motor nucleus of the vagus neurones to group II metabotropic glutamate receptor agonists in rats

    15 March 2014

    Recent studies have shown that pancreatic exocrine secretions (PES) are modulated by dorsal motor nucleus of the vagus (DMV) neurones, whose activity is finely tuned by GABAergic and glutamatergic synaptic inputs. Group II metabotropic glutamate receptors (mGluR) decrease synaptic transmission to pancreas-projecting DMV neurones and increase PES. In the present study, we used a combination of in vivo and in vitro approaches aimed at characterising the effects of caerulein-induced acute pancreatitis (AP) on the vagal neurocircuitry modulating pancreatic functions. In control rats, microinjection of bicuculline into the DMV increased PES, whereas microinjections of kynurenic acid had no effect. Conversely, in AP rats, microinjection of bicuculline had no effect, whereas kynurenic acid decreased PES. DMV microinjections of the group II mGluR agonist APDC and whole cell recordings of excitatory currents in identified pancreas-projecting DMV neurones showed a reduced functional response in AP rats compared to controls. Moreover, these changes persisted up to 3 weeks following the induction of AP. These data demonstrate that AP increases the excitatory input to pancreas-projecting DMV neurones by decreasing the response of excitatory synaptic terminals to group II mGluR agonist.

    Restless cell syndrome

    15 March 2014

    A novel computational model of mouse myocyte electrophysiology to assess the synergy between Na+ loading and CaMKII

    15 March 2014

    Ca2+–calmodulin-dependent protein kinase II (CaMKII) hyperactivity in heart failure causes intracellular Na+ ([Na+]i) loading (at least in part by enhancing the late Na+ current). This [Na+]i gain promotes intracellular Ca2+ ([Ca2+]i) overload by altering the equilibrium of the Na+–Ca2+ exchanger to impair forward-mode (Ca2+ extrusion), and favour reverse-mode (Ca2+ influx) exchange. In turn, this Ca2+ overload would be expected to further activate CaMKII and thereby form a pathological positive feedback loop of ever-increasing CaMKII activity, [Na+]i, and [Ca2+]i. We developed an ionic model of the mouse ventricular myocyte to interrogate this potentially arrhythmogenic positive feedback in both control conditions and when CaMKIIC is overexpressed as in genetically engineered mice. In control conditions, simulation of increased [Na+]i causes the expected increases in [Ca2+]i, CaMKII activity, and target phosphorylation, which degenerate into unstable Ca2+ handling and electrophysiology at high [Na+]i gain. Notably, clamping CaMKII activity to basal levels ameliorates but does not completely offset this outcome, suggesting that the increase in [Ca2+]i per se plays an important role. The effect of this CaMKII–Na+–Ca2+–CaMKII feedback is more striking in CaMKIIC overexpression, where high [Na+]i causes delayed afterdepolarizations, which can be prevented by imposing low [Na+]i, or clamping CaMKII phosphorylation of L-type Ca2+ channels, ryanodine receptors and phospholamban to basal levels. In this setting, Na+ loading fuels a vicious loop whereby increased CaMKII activation perturbs Ca2+ and membrane potential homeostasis. High [Na+]i is also required to produce instability when CaMKII is further activated by increased Ca2+ loading due to β-adrenergic activation. Our results support recent experimental findings of a synergistic interaction between perturbed Na+ fluxes and CaMKII, and suggest that pharmacological inhibition of intracellular Na+ loading can contribute to normalizing Ca2+ and membrane potential dynamics in heart failure.

    Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel-containing membrane microdomains

    15 March 2014

    Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that β-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

    Conduit artery structure and function in lowlanders and native highlanders: relationships with oxidative stress and role of sympathoexcitation

    01 March 2014

    Research detailing the normal vascular adaptions to high altitude is minimal and often confounded by pathology (e.g. chronic mountain sickness) and methodological issues. We examined vascular function and structure in: (1) healthy lowlanders during acute hypoxia and prolonged (~2 weeks) exposure to high altitude, and (2) high-altitude natives at 5050 m (highlanders). In 12 healthy lowlanders (aged 32 ± 7 years) and 12 highlanders (Sherpa; 33 ± 14 years) we assessed brachial endothelium-dependent flow-mediated dilatation (FMD), endothelium-independent dilatation (via glyceryl trinitrate; GTN), common carotid intima–media thickness (CIMT) and diameter (ultrasound), and arterial stiffness via pulse wave velocity (PWV; applanation tonometry). Cephalic venous biomarkers of free radical-mediated lipid peroxidation (lipid hydroperoxides, LOOH), nitrite (NO2–) and lipid soluble antioxidants were also obtained at rest. In lowlanders, measurements were performed at sea level (334 m) and between days 3–4 (acute high altitude) and 12–14 (chronic high altitude) following arrival to 5050 m. Highlanders were assessed once at 5050 m. Compared with sea level, acute high altitude reduced lowlanders’ FMD (7.9 ± 0.4 vs. 6.8 ± 0.4%; P = 0.004) and GTN-induced dilatation (16.6 ± 0.9 vs. 14.5 ± 0.8%; P = 0.006), and raised central PWV (6.0 ± 0.2 vs. 6.6 ± 0.3 m s–1; P = 0.001). These changes persisted at days 12–14, and after allometrically scaling FMD to adjust for altered baseline diameter. Compared to lowlanders at sea level and high altitude, highlanders had a lower carotid wall:lumen ratio (~19%, P ≤ 0.04), attributable to a narrower CIMT and wider lumen. Although both LOOH and NO2– increased with high altitude in lowlanders, only LOOH correlated with the reduction in GTN-induced dilatation evident during acute (n = 11, r = –0.53) and chronic (n = 7, r = –0.69; P ≤ 0.01) exposure to 5050 m. In a follow-up, placebo-controlled experiment (n = 11 healthy lowlanders) conducted in a normobaric hypoxic chamber (inspired O2 fraction (F IO 2) = 0.11; 6 h), a sustained reduction in FMD was evident within 1 h of hypoxic exposure when compared to normoxic baseline (5.7 ± 1.6 vs. 8.0 ±1.3%; P < 0.01); this decline in FMD was largely reversed following α1-adrenoreceptor blockade. In conclusion, high-altitude exposure in lowlanders caused persistent impairment in vascular function, which was mediated partially via oxidative stress and sympathoexcitation. Although a lifetime of high-altitude exposure neither intensifies nor attenuates the impairments seen with short-term exposure, chronic high-altitude exposure appears to be associated with arterial remodelling.

    Effects of natriuretic peptides on electrical conduction in the sinoatrial node and atrial myocardium of the heart

    01 March 2014

    Natriuretic peptides, including B-type and C-type natriuretic peptide (BNP and CNP), are powerful regulators of the cardiovascular system; however, their electrophysiological effects in the heart, particularly in the sinoatrial node (SAN), are incompletely understood. We have used high-resolution optical mapping to measure the effects of BNP and CNP, and the roles of natriuretic peptide receptors (NPR-A, NPR-B and NPR-C), on electrical conduction within the SAN and atrial myocardium. In basal conditions BNP and CNP (50–500 nm) increased conduction velocity (CV) within the SAN by ~30% at the high dose and shifted the initial exit site superiorly. These effects sped conduction from the SAN to the surrounding atrial myocardium and were mediated by the NPR-A and NPR-B receptors. In the presence of isoproterenol (1 μm) the NPR-C receptor made a major contribution to the effects of BNP and CNP in the heart. In these conditions BNP, CNP and the NPR-C agonist cANF each decreased SAN CV and shifted the initial exit site inferiorly. The effects of cANF (30% reduction) were larger than BNP or CNP (~15% reduction), indicating that BNP and CNP activate multiple natriuretic peptide receptors. In support of this, the inhibitory effects of BNP were absent in NPR-C knockout mice, where BNP instead elicited a further increase (~25%) in CV. Measurements in externally paced atrial preparations demonstrate that the effects of natriuretic peptides on CV are partially independent of changes in cycle length. These data provide detailed novel insight into the complex effects of natriuretic peptides and their receptors on electrical conduction in the heart.