Isolated Rat Soleus Research Articles

EXPRESSION PATTERN OF BK CHANNELS ON VARIOUS OXIDATIVE STRESS CONDITIONS IN SKELETAL MUSCLE

BK (large conductance Ca2+-activated potassium) channels are expressed in myocytes though changes in their molecular levels in the presence of oxidative stress is not clear, yet. Excessive production of reactive oxygen species leads to many diseases including periodic paralysis. Therefore, determination the molecular effects of various oxidative stress conditions may reveal the possible mechanism and potential therapeutic effects. In the present study, isolated rat soleus muscle where KCNMA1 genes encoding BK channel protein expressed widely in skeletal muscle, were exposed to cyclopiazonic acid (CPA) and also hydrogen peroxide (H2O2) as oxidative stress inducers. Streptozotocin-induced diabetes mellitus model was also used to demonstrate the effects of the endogenous source of oxidative stress. Moreover, NS1619, a BK channel opener was used whether the activation of the channel re-regulate the channel expression back. After the incubation periods, KCNMA1 gene expression levels of each groups were determined by real-time PCR experiments. While CPA and H2O2 decreased the KCNMA1 expression significantly, its expression did not change in systemic diabetes mellitus condition. However, the transcriptional level significantly decreased in diabetes in the presence of H2O2. On the other hand, KCNMA1 expression was re-regulated back to the control’s level by addition of NS1619 in solely hydrogen peroxide groups. The results demonstrated for the first time that acute oxidative stress, rather than systemic conditions, effects the KCNMA1 gene expression level in skeletal muscle. The study was also showed the effects of NS1619 on the regulation of transcriptional levels of BK channel protein in hydrogen peroxide conditions.

Potassium-induced potentiation of subtetanic force in rat skeletal muscles: influences of β2-activation, lactic acid, and temperature.

Moderate elevations of extracellular K+ concentration ([K+]o) occur during exercise and have been shown to potentiate force during contractions elicited with subtetanic frequencies. Here, we investigated whether lactic acid (reduced chloride conductance), β2-adrenoceptor activation, and increased temperature would influence the potentiating effect of potassium in slow- and fast-twitch muscles. Isometric contractions were elicited by electrical stimulation at various frequencies in isolated rat soleus and extensor digitorum longus (EDL) muscles incubated at normal (4 mM) or elevated K+, in combination with salbutamol (5 μM), lactic acid (18.1 mM), 9-anthracene-carboxylic acid (9-AC; 25 μM), or increased temperature (30-35°C). Elevating [K+]o from 4 mM to 7 mM (soleus) and 10 mM (EDL) potentiated isometric twitch and subtetanic force while slightly reducing tetanic force. In EDL, salbutamol further augmented twitch force (+27 ± 3%, P < 0.001) and subtetanic force (+22 ± 4%, P < 0.001). In contrast, salbutamol reduced subtetanic force (-28 ± 6%, P < 0.001) in soleus muscles. Lactic acid and 9-AC had no significant effects on isometric force of muscles already exposed to moderate elevations of [K+]o. The potentiating effect of elevated [K+]o was still well maintained at 35°C. Addition of salbutamol exerts a further force-potentiating effect in fast-twitch but not in slow-twitch muscles already potentiated by moderately elevated [K+]o, whereas lactic acid, 9-AC, or increased temperature does not exert any further augmentation. However, the potentiating effect of elevated [K+]o was still maintained in the presence of these, thus emphasizing the positive influence of moderately elevated [K+]o for contractile performance during exercise.

The Role of Stretch-Activated Channels in Mediating mTORC1 Signaling in Isolated Rat Soleus Muscle in Response to Mechanical Stimulation after Functional Unloading

The aim of the present work was to identify the role of stretch-activated ion channels (SAC) in the transmission of mechanical signals to the key mTORC1/p70S6k anabolic pathways in the soleus muscle in rats after seven days of functional unloading of the hindlimbs. Functional unloading of the hindlimb muscles was by antiorthostatic suspension (hindlimb suspension, HS). The rats were divided into four groups: 1) group C (animal-house control; 2) C + Gd (controls whose muscles were incubated with the SAC inhibitor GdCl3; 3) HS (functional unloading for seven days); and 4) HS + Gd (a group with seven days of antiorthostatic suspension followed by incubation of muscles with GdCl3). After completion of antiorthostatic suspension, isolated soleus muscles were subjected to a series of eccentric contractions (EC). Muscles from intact animals responded to EC with increases in the phosphorylation of key markers of mTORC1 signaling: p70S6k and rpS6. The extent of mechanically induced increases in the levels of phosphorylation of these protein markers were significantly reduced to essentially identical extents in the “pure” suspension (HS) group and the HS group given the SAC blocker (HS + Gd) as compared with the increase in muscles from intact control group. These results lead to the conclusion that decreases in the mechanically dependent responses of the mTORC1 signal pathway in the soleus muscle in rats after seven days of functional unloading may be linked with inactivation of stretch-activated ion channels.

Is Fatigue-induced Loss Of Power Associated With a Change In The Curvature Of The Force-velocity Relation?

In skeletal muscles, the ability to generate power is reduced during fatigue. Maximal power (Pmax) is determined by the force-velocity relationship, which can be closely approximated by the Hill equation, containing three key parameters: 1) Maximal isometric force (Fmax) 2) Maximum contraction velocity (Vmax) and 3) Force-velocity curvature. PURPOSE: To investigate the possible association between the fatigue-induced loss of power and changes in curvature of the force-velocity relation in muscles with different fiber type composition. METHODS: The force-velocity relationship was measured before and during development of fatigue in isolated rat soleus (slow-twitch) and EDL (fast-twitch). Muscles were incubated in Krebs-Ringer solution at 30 C° and stimulated electrically at 60 Hz for 0.75 s (soleus) or at 150 Hz for 0.2 s (EDL) to obtain serial concentric contractions leading to fatigue. The Force-velocity relationship was measured at different stages of fatigue and after 1 hour of recovery. Force-velocity data were fitted to the Hill equation, and curvature was determined as the ratio of the curve parameters a/F0 (inversely related to curvature). RESULTS: At the end of the fatiguing protocol, maximal power had decreased by 58 ± 5% (soleus) and 69 ± 4% (EDL) compared to initial values in non-fatigued muscles. Curvature increased in both muscle types as judged from the decrease in a/F0 of 81 ± 20% (soleus) and by 31 ± 12% (EDL). At the end of the fatiguing protocol, the calculated contributions to the total loss of Pmax was 25 ± 10% from Fmax, 17 ± 3% from Vmax and 58 ± 14% from a/F0 (soleus) and 64 ± 6% from Fmax, 16 ± 3% from Vmax and 20 ± 9% from a/F0 (EDL). Complete recovery of a/F0 was observed following one hour of rest in both muscle types. CONCLUSIONS: Increased curvature of the force-velocity relationship occurs during fatigue in both slow and fast twitch muscles. Particularly in slow-twitch muscles, this increase in curvature is strongly associated with fatigue-induced loss of power.

Fatiguing stimulation increases curvature of the force-velocity relationship in isolated fast-twitch and slow-twitch rat muscles.

In skeletal muscles, the ability to generate power is reduced during fatigue. In isolated muscles, maximal power can be calculated from the force-velocity relationship. This relationship is well described by the Hill equation, which contains three parameters: (1) maximal isometric force, (2) maximum contraction velocity and (3) curvature. Here, we investigated the hypothesis that a fatigue-induced loss of power is associated with changes in curvature of the force-velocity curve in slow-twitch muscles but not in fast-twitch muscles during the development of fatigue. Isolated rat soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles were incubated in Krebs-Ringer solution at 30°C and stimulated electrically at 60 Hz (soleus) and 150 Hz (EDL) to perform a series of concentric contractions to fatigue. Force-velocity data were fitted to the Hill equation, and curvature was determined as the ratio of the curve parameters a/F0 (inversely related to curvature). At the end of the fatiguing protocol, maximal power decreased by 58±5% in the soleus and 69±4% in the EDL compared with initial values in non-fatigued muscles. At the end of the fatiguing sequence, curvature increased as judged from the decrease in a/F0 by 81±20% in the soleus and by 31±12% in the EDL. However, during the initial phases of fatiguing stimulation, we observed a small decrease in curvature in the EDL, but not in the soleus, which may be a result of post-activation potentiation. In conclusion, fatigue-induced loss of power is strongly associated with an increased curvature of the force-velocity relationship, particularly in slow-twitch muscles.

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading.&#x0D; Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique.&#x0D; Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS.&#x0D; Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055.

ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK

ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK

Effects of manipulating tetanic calcium on the curvature of the force-velocity relationship in isolated rat soleus muscles.

In dynamically contracting muscles, increased curvature of the force-velocity relationship contributes to the loss of power during fatigue. It has been proposed that fatigue-induced reduction in [Ca++ ]i causes this increased curvature. However, earlier studies on single fibres have been conducted at low temperatures. Here, we investigated the hypothesis that curvature is increased by reductions in tetanic [Ca++ ]i in isolated skeletal muscle at near-physiological temperatures. Rat soleus muscles were stimulated at 60Hz in standard Krebs-Ringer buffer, and contraction force and velocity were measured. Tetanic [Ca++ ]i was in some experiments either lowered by addition of 10μmol/L dantrolene or use of submaximal stimulation (30Hz) or increased by addition of 2mmol/L caffeine. Force-velocity curves were constructed by fitting shortening velocity at different loading forces to the Hill equation. Curvature was determined as the ratio a/F0 with increased curvature reflecting decreased a/F0 . Compared to control levels, lowering tetanic [Ca++ ]i with dantrolene or reduced stimulation frequency decreased the curvature slightly as judged from increase in a/F0 of 13±1% (P=<.001) and 20±2% (P=<.001) respectively. In contrast, increasing tetanic [Ca++ ]i with caffeine increased the curvature (a/F0 decreased by 17±1%; P=<.001). Contrary to our hypothesis, interventions that reduced tetanic [Ca++ ]i caused a decrease in curvature, while increasing tetanic [Ca++ ]i increased the curvature. These results reject a simple causal relation between [Ca++ ]i and curvature of the force-velocity relation during fatigue.

Effects of ATP and adenosine on contraction amplitude of rat soleus muscle at different temperatures.

The aim of this study was to evaluate the effects of adenosine 5'-triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30-14°C) and recorded in the presence of ATP, adenosine, suramin, and 8-(p-sulfophenyl)-theophylline (8-SPT). At 37°C, incubation of the muscles with ATP inhibited ES-induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES-induced contractions at all temperature levels; 8-SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol-induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. ATP is involved in both pre- and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55: 417-423, 2017.

The realization of a mechanical signal during gravitational unloading: The response of mTORC1 targets to eccentric contractions

The aim of this study was to assess the response of key mTORC1 substrates to a bout of contractile stimuli under different times of functional unloading. Functional unloading of hind-limb muscles was carried out by the method of antiorthostatic suspension. Twenty-eight Wistar rats were divided into four groups: control, and hindlimb suspension for 1, 3, and 7 days. After hindlimb suspension, isolated soleus muscles of rats were subjected to a bout of ex vivo eccentric contractions. The contents of phosphorylated forms of p70s6k and 4E-BP1 were then determined using western blotting. It was found that an eccentric load resulted in a significant increase in p70s6k phosphorylation and reduced 4E-BP1 phosphorylation both in control and suspended rats, but in the case of suspension the response was dramatically reduced. Thus, it can be concluded that a bout of eccentric contractions of isolated rat soleus muscle during functional unloading causes a weaker activation of the Akt-mTORC1-p70s6k signaling pathway compared with the control. This may indicate that it is important to maintain muscle tone for a more efficient muscle perception of an external mechanical signal and subsequent activation of anabolic signaling pathways.

Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle.

Calorie restriction (CR; ∼60%-65% of ad libitum consumption) can enhance insulin-stimulated glucose uptake (ISGU) in predominantly slow-twitch skeletal muscles (e.g., soleus) by an incompletely understood mechanism. We used an Akt inhibitor (MK-2206) to eliminate CR's effect on insulin-stimulated Akt2 phosphorylation in isolated rat soleus muscles. We found long-term CR-enhanced ISGU was abolished by eliminating the CR effect on Akt2 phosphorylation, suggesting the CR-induced benefit on ISGU in the predominantly slow-twitch soleus relies on enhanced Akt2 phosphorylation.

Muscle Fatigue Affects the Interpolated Twitch Technique When Assessed Using Electrically-Induced Contractions in Human and Rat Muscles.

The interpolated twitch technique (ITT) is the gold standard to assess voluntary activation and central fatigue. Yet, its validity has been questioned. Here we studied how peripheral fatigue can affect the ITT. Repeated contractions at submaximal frequencies were produced by supramaximal electrical stimulations of the human adductor pollicis muscle in vivo and of isolated rat soleus fiber bundles; an extra stimulation pulse was given during contractions to induce a superimposed twitch. Human muscles fatigued by repeated 30-Hz stimulation trains (3 s on–1 s off) showed an ~80% reduction in the superimposed twitch force accompanied by a severely reduced EMG response (M-wave amplitude), which implies action potential failure. Subsequent experiments combined a less intense stimulation protocol (1.5 s on–3 s off) with ischemia to cause muscle fatigue, but which preserved M-wave amplitude. However, the superimposed twitch force still decreased markedly more than the potentiated twitch force; with ITT this would reflect increased “voluntary activation.” In contrast, the superimposed twitch force was relatively spared when a similar protocol was performed in rat soleus bundles. Force relaxation was slowed by >150% in fatigued human muscles, whereas it was unchanged in rat soleus bundles. Accordingly, results similar to those in the human muscle were obtained when relaxation was slowed by cooling the rat soleus muscles. In conclusion, our data demonstrate that muscle fatigue can confound the quantification of central fatigue using the ITT.

Gender-specific neuromuscular adaptations to unloading in isolated rat soleus muscles.

The potential of gender to affect unloading-induced neuromuscular adaptations was investigated. Twenty male and 20 female rats were assigned to control (CTL), or unloaded (UL) conditions. After 2 weeks of unloading, soleus muscles were removed, and neuromuscular function was assessed during a train of alternating indirect (neural) and direct (muscle) stimuli. In rested muscle, strength showed significant (P ≤ 0.05) main effects for gender (male > female) and treatment (CTL > UL). By the end of the testing protocol, when muscles showed fatigue, gender-related and treatment-related differences in strength had disappeared. Neuromuscular transmission efficiency and strength suffered a greater decline during the testing protocol in males than females. Unloaded male muscles displayed greater contractile velocity than female muscles both when rested and fatigued. Gender affected unloading-induced neuromuscular adaptations. The greater strength of rested male muscles was due to greater muscle mass and neuromuscular transmission efficiency. Muscle Nerve 54: 300-307, 2016.

Positional differences in reactive hyperemia provide insight into initial phase of exercise hyperemia.

Studies have reported a greater blood flow response to muscle contractions when the limb is below the heart compared with above the heart, and these results have been interpreted as evidence for a skeletal muscle pump contribution to exercise hyperemia. If limb position affects the blood flow response to other vascular challenges such as reactive hyperemia, this interpretation may not be correct. We hypothesized that the magnitude of reactive hyperemia would be greater with the limb below the heart. Brachial artery blood flow (Doppler ultrasound) and blood pressure (finger-cuff plethysmography) were measured in 10 healthy volunteers. Subjects lay supine with one arm supported in two different positions: above or below the heart. Reactive hyperemia was produced by occlusion of arterial inflow for varying durations: 0.5 min, 1 min, 2 min, or 5 min in randomized order. Peak increases in blood flow were 77 ± 11, 178 ± 24, 291 ± 25, and 398 ± 33 ml/min above the heart and 96 ± 19, 279 ± 62, 550 ± 60, and 711 ± 69 ml/min below the heart (P < 0.05). Thus a standard stimulus (vascular occlusion) elicited different responses depending on limb position. To determine whether these differences were due to mechanisms intrinsic to the arterial wall, a second set of experiments was performed in which acute intraluminal pressure reduction for 0.5 min, 1 min, 2 min, or 5 min was performed in isolated rat soleus feed arteries (n = 12). The magnitude of dilation upon pressure restoration was greater when acute pressure reduction occurred from 85 mmHg (mimicking pressure in the arm below the heart; 28.3 ± 7.9, 37.5 ± 5.9, 55.1 ± 9.9, and 68.9 ± 8.6% dilation) than from 48 mmHg (mimicking pressure in the arm above the heart; 20.8 ± 4.8, 22.6 ± 4.4, 31.2 ± 5.8, and 49.2 ± 7.1% dilation). These data support the hypothesis that arm position differences in reactive hyperemia are at least partially mediated by mechanisms intrinsic to the arterial wall. Overall, these results suggest the need to reevaluate studies employing positional changes to examine muscle pump influences on exercise hyperemia.

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Critical role of the transient activation of p38 MAPK in the etiology of skeletal muscle insulin resistance induced by low-level in vitro oxidant stress

Increased cellular exposure to oxidants may contribute to the development of insulin resistance and type 2 diabetes. Skeletal muscle is the primary site of insulin-dependent glucose disposal in the body; however, the effects of oxidative stress on insulin signaling and glucose transport activity in mammalian skeletal muscle are not well understood. We therefore studied the effects of a low-level in vitro oxidant stress (30–40 μM H 2O 2) on basal and insulin-stimulated (5 mU/ml) glucose transport activity and insulin signaling at 2, 4, and 6 h in isolated rat soleus muscle. H 2O 2 increased basal glucose transport activity at 2 and 4 h, but not at 6 h. This low-level oxidant stress significantly impaired insulin-stimulated glucose transport activity at all time points, and was associated with inhibition of insulin-stimulated phosphorylation of Akt Ser 473 and GSK-3β Ser 9. In the presence of insulin, H 2O 2 decreased total protein expression of IRS-1 at 6 h and IRS-2 at 4 and 6 h. Phosphorylation of p38 MAPK Thr 180/Tyr 182 was transiently increased by H 2O 2 in the presence and absence of insulin at 2 and 4 h, but not at 6 h. Selective inhibition of p38 MAPK with A304000 partially rescued the H 2O 2-induced reduction in insulin-stimulated glucose transport activity. These results indicate that direct in vitro exposure of isolated mammalian skeletal muscle to a low-level oxidant stress impairs distal insulin signaling and insulin-stimulated glucose transport activity, at least in part, due to a p38 MAPK-dependent mechanism.

A novel two-chain IGF-II-derived peptide from purified β-cell granules

Objective Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic β-cells. Here we isolated secretory granules from a β-cell line, βTC6-F7, and characterized the nature of the IGF-II-LI located therein. Design Secretory granules were isolated from cultured mouse βTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH 2-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. Results βTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 10 6 cells compared to 340 ± 44 pmol insulin-LI per 10 6 cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37–40)IGF-II, which we termed ‘vesiculin’. This molecule was also detectable in βTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC 50 value of 131 nM ± 1.35. Conclusions Vesiculin, des(37–40)IGF-II, is a novel two-chain insulin-like hormone and the major “IGF-II-like” peptide found in purified mouse βTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species.

Berberine-induced activation of 5′-adenosine monophosphate–activated protein kinase and glucose transport in rat skeletal muscles

Berberine (BBR) is the main alkaloid of Coptis chinensis, which has been used as a folk medicine to treat diabetes mellitus in Asian countries. We explored the possibility that 5′-adenosine monophosphate–activated protein kinase (AMPK) is involved in metabolic enhancement by BBR in skeletal muscle, the important tissue for glucose metabolism. Isolated rat epitrochlearis and soleus muscles were incubated in a buffer containing BBR, and activation of AMPK and related events were examined. In response to BBR treatment, the Thr 172 phosphorylation of the catalytic α-subunit of AMPK, an essential step for full kinase activation, increased in a dose- and time-dependent manner. Ser 79 phosphorylation of acetyl–coenzyme A carboxylase, an intracellular substrate of AMPK, increased correspondingly. Analysis of isoform-specific AMPK activity revealed that BBR activated both the α1 and α2 isoforms of the catalytic subunit. This increase in enzyme activity was associated with an increased rate of 3- O-methyl- d-glucose transport in the absence of insulin and with phosphorylation of AS160, a signaling intermediary leading to glucose transporter 4 translocation. The intracellular energy status estimated from the phosphocreatine concentration was decreased by BBR. These results suggest that BBR acutely stimulates both AMPK α1 and AMPK α2 and insulin-independent glucose transport in skeletal muscle with a reduction of the intracellular energy status.

Prolonged AICAR-induced AMP-kinase activation promotes energy dissipation in white adipocytes: novel mechanisms integrating HSL and ATGL

This study was designed to investigate the effects of prolonged activation of AMP-activated protein kinase (AMPK) on lipid partitioning and the potential molecular mechanisms involved in these processes in white adipose tissue (WAT). Rat epididymal adipocytes were incubated with 5'-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside (AICAR;0.5 mM) for 15 h. Also, epididymal adipocytes were isolated 15 h after AICAR was injected (i.p. 0.7 g/kg body weight) in rats. Adipocytes were utilized for various metabolic assays and for determination of gene expression and protein content. Time-dependent in vivo plasma NEFA concentrations were determined. AICAR treatment significantly increased AMPK activation, inhibited lipogenesis, and increased FA oxidation. This was accompanied by upregulation of peroxisome proliferator-activated receptor (PPAR)alpha, PPARdelta, and PPARgamma-coactivator-1alpha (PGC-1alpha) mRNA levels. Lipolysis was first suppressed, but then increased, both in vitro and in vivo, with prolonged AICAR treatment. Exposure to AICAR increased adipose triglyceride lipase (ATGL) content and FA release, despite inhibition of basal and epinephrine-stimulated hormone-sensitive lipase (HSL) activity. Here, we provide evidence that prolonged AICAR-induced AMPK activation can remodel adipocyte metabolism by upregulating pathways that favor energy dissipation versus lipid storage in WAT. Additionally, we show novel time-dependent effects of AICAR-induced AMPK activation on lipolysis, which involves antagonistic modulation of HSL and ATGL.

Resistin acutely impairs insulin-stimulated glucose transport in rodent muscle in the presence, but not absence, of palmitate

Resistin is a cytokine implicated in the development of insulin resistance. However, there has been little investigation of the effects of resistin on fatty acid (FA) metabolism and insulin response in skeletal muscle, a key tissue for glucose disposal. The purpose of the present study was to examine the role of altered FA metabolism as a cause of resistin's inhibition of insulin-stimulated glucose transport in muscle. Isolated rat soleus muscles were incubated acutely (2 h) in the presence or absence of 600 ng/ml resistin, with or without 2 mM palmitate. Resistin acutely impaired insulin-stimulated glucose transport and Akt phosphorylation, but only in the presence of palmitate, implicating a role for altered FA metabolism. This impairment of glucose transport induced by resistin plus palmitate could be pharmacologically rescued by the inclusion of aimidazole carboxamide ribonucleotide, a stimulator of AMP-activated protein kinase and FA oxidation, as well as inhibitors of ceramide synthesis (myriocin, fumonisin). However, to our surprise, resistin actually blunted the palmitate-induced increase in muscle ceramide content; as expected, ceramide content was significantly lowered by fumonisin. In summary, the acute impairment of insulin response by resistin was manifested only in the presence of high palmitate and was alleviated when FA metabolism was manipulated (increased oxidation, inhibited ceramide synthesis). Resistin's acute impairment of insulin response does not appear to require an absolute increase in ceramide content; however, reducing ceramide content alleviated the impairment in glucose transport and insulin signaling.