Alexander V. Chibalin

Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans

Skeletal muscle from strength‐ and endurance‐trained individuals represents diverse adaptive states. In this regard, AMPK‐PGC‐1α signaling mediates several adaptations to endurance training, while up‐regulation of the Akt‐TSC2‐mTOR pathway may underlie increased protein synthesis after resistance exercise. We determined the effect of prior training history on signaling responses in seven strength‐trained and six endurance‐trained males who undertook 1 h cycling at 70% VO2peak or eight sets of five maximal repetitions of isokinetic leg extensions. Muscle biopsies were taken at rest, immediately and 3 h postexercise. AMPK phosphorylation increased after cycling in strength‐trained (54%; P<0.05) but not endurance‐trained subjects. Conversely, AMPK was elevated after resistance exercise in endurance‐ (114%; P<0.05), but not strength‐trained subjects. Akt phosphorylation increased in endurance‐ (50%; P<0.05), but not strength‐trained subjects after cycling but was unchanged in either group after resistance exercise. TSC2 phosphorylation was decreased (47%; P<0.05) in endurance‐trained subjects following resistance exercise, but cycling had little effect on the phosphorylation state of this protein in either group. p70S6K phosphorylation increased in endurance‐ (118%; P<0.05), but not strength‐trained subjects after resistance exercise, but was similar to rest in both groups after cycling. Similarly, phosphorylation of S6 protein, a substrate for p70 S6K, was increased immediately following resistance exercise in endurance‐ (129%; P<0.05), but not strength‐trained subjects. In conclusion, a degree of “response plasticity” is conserved at opposite ends of the endurance‐hypertrophic adaptation continuum. Moreover, prior training attenuates the exercise specific signaling responses involved in single mode adaptations to training.

Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle

The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one‐leg cycle ergometry at ∼70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction‐induced signaling in human skeletal. Activation of the different MAP kinase pathways byphysical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.—Widegren, U., Jiang, X.‐J., Krook, A., Chibalin, A. V., Bjo¨rnholm, M., Tally, M., Roth, R. A., Henriksson, J., Wallberg‐ Henriksson, H., Zierath, J. R. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle. FASEB J. 12, 1379– 1389 (1998)

AMPK-Mediated AS160 Phosphorylation in Skeletal Muscle Is Dependent on AMPK Catalytic and Regulatory Subunits

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 β-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (α1β1γ1 and α2β2γ1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (α2 AMPK knockout [KO], α2 AMPK kinase dead [KD], and γ3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing α2 and γ3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in α2 AMPK KO and KD but not γ3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

Exercise intensity‐dependent regulation of peroxisome proliferator‐activated receptor γ coactivator‐1α mRNA abundance is associated with differential activation of upstream signalling kinases in human skeletal muscle

Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α)‐dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC‐1α expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC‐1α transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 ± 4.0 min; HI, 36.0 ± 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC‐1α mRNA abundance increased in an intensity‐dependent manner +3 h after exercise (LO, 3.8‐fold; HI, 10.2‐fold, P < 0.05). AMP‐activated protein kinase (AMPK) (2.8‐fold, P < 0.05) and calcium/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen‐activated protein kinase (MAPK) phosphorylation increased after both trials (∼2.0‐fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor‐2 (ATF‐2), increased only after HI (2.4‐fold, P < 0.05). Cyclic‐AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (∼80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0‐fold, P < 0.05). In conclusion, exercise intensity regulates PGC‐1α mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of multiple signalling pathways, with ATF‐2 and HDAC phosphorylation proposed as key intensity‐dependent mediators.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic β cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the β cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis.

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its α- and β-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the α-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the α-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent α-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the α-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent α-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the α-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Metabolic and mitogenic signal transduction in human skeletal muscle after intense cycling exercise

We determined whether mitogen‐activated protein kinase (MAPK) and 5′‐AMP‐activated protein kinase (AMPK) signalling cascades are activated in response to intense exercise in skeletal muscle from six highly trained cyclists (peak O2 uptake (V̇O2,peak) 5.14 ± 0.1 l min−1) and four control subjects (V̇O2,peak 3.8 ± 0.1 l min−1) matched for age and body mass. Trained subjects completed eight 5 min bouts of cycling at ≈85% of V̇O2,peak with 60 s recovery between work bouts. Control subjects performed four 5 min work bouts commencing at the same relative, but a lower absolute intensity, with a comparable rest interval. Vastus lateralis muscle biopsies were taken at rest and immediately after exercise. Extracellular regulated kinase (ERK1/2), p38 MAPK, histone H3, AMPK and acetyl CoA‐carboxylase (ACC) phosphorylation was determined by immunoblot analysis using phosphospecific antibodies. Activity of mitogen and stress‐activated kinase 1 (MSK1; a substrate of ERK1/2 and p38 MAPK) and α1 and α2 subunits of AMPK were determined by immune complex assay. ERK1/2 and p38 MAPK phosphorylation and MSK1 activity increased (P < 0.05) after exercise 2.6‐, 2.1‐ and 2.0‐fold, respectively, in control subjects and 1.5‐, 1.6‐ and 1.4‐fold, respectively, in trained subjects. Phosphorylation of histone H3, a substrate of MSK1, increased (P < 0.05) ≈1.8‐fold in both control and trained subject. AMPKα2 activity increased (P < 0.05) after exercise 4.2‐ and 2.3‐fold in control and trained subjects, respectively, whereas AMPKα1 activity was not altered. Exercise increased ACC phosphorylation (P < 0.05) 1.9‐ and 2.8‐fold in control and trained subjects. In conclusion, intense cycling exercise in subjects with a prolonged history of endurance training increases MAPK signalling to the downstream targets MSK1 and histone H3 and isoform‐specific AMPK signalling to ACC. Importantly, exercise‐induced signalling responses were greater in untrained men, even at the same relative exercise intensity, suggesting muscle from previously well‐trained individuals requires a greater stimulus to activate signal transduction via these pathways.

Muscle fiber type specificity in insulin signal transduction

We determined the muscle fiber type-specific response of intracellular signaling proteins to insulin. Epitrochlearis (Epi; 15% type I, 20% type IIa, and 65% type IIb), soleus (84, 16, and 0%), and extensor digitorum longus (EDL; 3, 57, and 40%) muscles from Wistar rats were incubated without or with 120 nM insulin (3-40 min). Peak insulin receptor (IR) tyrosine phosphorylation was reached after 6 (soleus) and 20 (Epi and EDL) min, with sustained activity throughout insulin exposure (40 min). Insulin increased insulin receptor substrate (IRS)-1 and IRS-2 tyrosine phosphorylation and phosphotyrosine-associated phosphatidylinositol (PI)-3-kinase activity to a maximal level after 3-10 min, with subsequent downregulation. Akt kinase phosphorylation peaked at 20 min, with sustained activity throughout insulin exposure. Importantly, the greatest insulin response for all signaling intermediates was observed in soleus, whereas the insulin response between EDL and Epi was similar. Protein expression of the p85α-subunit of PI 3-kinase and Akt kinase, but not IR, IRS-1, or IRS-2, was greater in oxidative versus glycolytic muscle. In conclusion, increased function and/or expression of key proteins in the insulin-signaling cascade contribute to fiber type-specific differences in insulin action in skeletal muscle.We determined the muscle fiber type-specific response of intracellular signaling proteins to insulin. Epitrochlearis (Epi; 15% type I, 20% type IIa, and 65% type IIb), soleus (84, 16, and 0%), and extensor digitorum longus (EDL; 3, 57, and 40%) muscles from Wistar rats were incubated without or with 120 nM insulin (3-40 min). Peak insulin receptor (IR) tyrosine phosphorylation was reached after 6 (soleus) and 20 (Epi and EDL) min, with sustained activity throughout insulin exposure (40 min). Insulin increased insulin receptor substrate (IRS)-1 and IRS-2 tyrosine phosphorylation and phosphotyrosine-associated phosphatidylinositol (PI)-3-kinase activity to a maximal level after 3-10 min, with subsequent downregulation. Akt kinase phosphorylation peaked at 20 min, with sustained activity throughout insulin exposure. Importantly, the greatest insulin response for all signaling intermediates was observed in soleus, whereas the insulin response between EDL and Epi was similar. Protein expression of the p85alpha-subunit of PI 3-kinase and Akt kinase, but not IR, IRS-1, or IRS-2, was greater in oxidative versus glycolytic muscle. In conclusion, increased function and/or expression of key proteins in the insulin-signaling cascade contribute to fiber type-specific differences in insulin action in skeletal muscle.

Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of α-subunits in lung alveolar cells

Catecholamines promote lung edema clearance via β-adrenergic-mediated stimulation of active Na+ transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+ flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to β-adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 μM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 μM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase α-subunit; rather, it was the result of an increased number of α-subunits recruited from the late endosomes into the plasma membrane. The recruitment of α-subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting α-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.