Stine J. Maarbjerg

AMP-activated protein kinase (AMPK) β1β2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise

AMP-activated protein kinase (AMPK) β1 or β2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle, α2β2γ3-containing heterotrimers predominate. However, compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK α2, β2, and γ3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism, because these models have normal mitochondrial content, contraction-stimulated glucose uptake, and insulin sensitivity. In the current study, we generated mice lacking both AMPK β1 and β2 isoforms in skeletal muscle (β1β2M-KO). β1β2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly, young β1β2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake, findings that were not apparent in mice with single mutations or deletions in muscle α, β, or γ subunits.

Current understanding of increased insulin sensitivity after exercise - emerging candidates.

Exercise counteracts insulin resistance and improves glucose homeostasis in many ways. Apart from increasing muscle glucose uptake quickly, exercise also clearly increases muscle insulin sensitivity in the post‐exercise period. This review will focus on the mechanisms responsible for this increased insulin sensitivity. It is believed that increased sarcolemmal content of the glucose transporter GLUT4 can explain the phenomenon to some extent. Surprisingly no improvement in the proximal insulin signalling pathway is observed at the level of the insulin receptor, IRS1, PI3K or Akt. Recently more distal signalling component in the insulin signalling pathway such as aPKC, Rac1, TBC1D4 and TBC1D1 have been described. These are all affected by both insulin and exercise which means that they are likely converging points in promoting GLUT4 translocation and therefore possible candidates for regulating insulin sensitivity after exercise. Whereas TBC1D1 does not appear to regulate insulin sensitivity after exercise, correlative evidence in contrast suggests TBC1D4 to be a relevant candidate. Little is known about aPKC and Rac1 in relation to insulin sensitivity after exercise. Besides mechanisms involved in signalling to GLUT4 translocation, factors influencing the trans‐sarcolemmal glucose concentration gradient might also be important. With regard to the interstitial glucose concentration microvascular perfusion is particular relevant as correlative evidence supports a connection between insulin sensitivity and microvascular perfusion. Thus, there are new candidates at several levels which collectively might explain the phenomenon.

A Ca2+–calmodulin–eEF2K–eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions

Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesized that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E‐binding protein 1 (4EBP1) phosphorylation by signalling cascades downstream of rises in intracellular [Ca2+] and decreased energy charge via AMP‐activated protein kinase (AMPK) in contracting skeletal muscle. When fast‐twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis correlated more closely with changes in eEF2 than 4EBP1 phosphorylation. Using a combination of Ca2+ release agents and ATPase inhibitors it was shown that the 60–70% suppression of fast‐twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca2+ and energy turnover‐related mechanisms. Furthermore, eEF2 kinase (eEF2K) inhibition completely blunted increases in eEF2 phosphorylation and partially blunted (i.e. 30–40%) the suppression of protein synthesis during contractions. The 3‐ to 5‐fold increase in skeletal muscle eEF2 phosphorylation during contractions in situ was rapid and sustained and restricted to working muscle. The increase in eEF2 phosphorylation and eEF2K activation were downstream of Ca2+–calmodulin (CaM) but not other putative activating factors such as a fall in intracellular pH or phosphorylation by protein kinases. Furthermore, blunted protein synthesis and 4EBP1 dephosphorylation were unrelated to AMPK activity during contractions, which was exemplified by normal blunting of protein synthesis during contractions in muscles overexpressing kinase‐dead AMPK. In summary, in fast‐twitch skeletal muscle, the inhibition of eEF2 activity by phosphorylation downstream of Ca2+–CaM–eEF2K signalling partially contributes to the suppression of protein synthesis during exercise/contractions.

Rac1 Is a Novel Regulator of Contraction-Stimulated Glucose Uptake in Skeletal Muscle

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

Trigeminal Neuralgia – A Prospective Systematic Study of Clinical Characteristics in 158 Patients

To prospectively describe the clinical characteristics of classical trigeminal neuralgia (TN) in a standardized manner.

Significance of neurovascular contact in classical trigeminal neuralgia

Neurovascular contact is considered a frequent cause of classical trigeminal neuralgia and microvascular decompression with transposition of a blood vessel is preferred over other surgical options in medically refractory patients with classical trigeminal neuralgia. However, the prevalence of neurovascular contact has not been investigated in a representative cohort of patients with classical trigeminal neuralgia based in a neurological setting and using high-quality neuroimaging and blinded evaluation. We aimed to investigate whether presence and degree of neurovascular contact are correlated to pain side in classical trigeminal neuralgia. Consecutive classical trigeminal neuralgia patients with unilateral symptoms were referred to 3.0 T magnetic resonance imaging and included in a cross-sectional study. Magnetic resonance imaging scans were evaluated blindly and graded according to presence and degree of neurovascular contact. Severe neurovascular contact was defined as displacement or atrophy of the trigeminal nerve. A total of 135 patients with classical trigeminal neuralgia were included. Average age of disease onset was 53.0 years (95% confidence interval mean 40.5-55.5) and current age was 60.1 years (95% % confidence interval mean 57.5-62.7). Eighty-two (61%, 95% confidence interval 52-69%) patients were female. Neurovascular contact was prevalent both on the symptomatic and asymptomatic side [89% versus 78%, P = 0.014, odds ratio = 2.4 (1.2-4.8), P = 0.017], while severe neurovascular contact was highly prevalent on the symptomatic compared to the asymptomatic side [53% versus 13%, P < 0.001, odds ratio = 11.6 (4.7-28.9), P < 0.001]. Severe neurovascular contact was caused by arteries in 98%. We conclude that neurovascular contact causing displacement or atrophy of the trigeminal nerve is highly associated with the symptomatic side in classical trigeminal neuralgia as opposed to neurovascular contact in general. Our findings demonstrate that severe neurovascular contact is involved in the aetiology of classical trigeminal neuralgia and that it is caused by arteries located in the root entry zone.

LKB1 Regulates Lipid Oxidation During Exercise Independently of AMPK

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD+ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.

Differential effect of bicycling exercise intensity on activity and phosphorylation of atypical protein kinase C and extracellular signal‐regulated protein kinase in skeletal muscle

Atypical protein kinase C (aPKC) and extracellular signal‐regulated kinase (ERK) are emerging as important signalling molecules in the regulation of metabolism and gene expression in skeletal muscle. Exercise is known to increase activity of aPKC and ERK in skeletal muscle but the effect of exercise intensity hereon has not been studied. Furthermore, the relationship between activity and phosphorylation of the two enzymes during exercise is unknown. Nine healthy young men exercised for 30 min on a bicycle ergometer on two occasions. One occasion consisted of three consecutive 10 min bouts of 35, 60 and 85% of peak pulmonary oxygen uptake and the second of one 30 min bout at 35% of . Both trials also included 30 min recovery. Muscle biopsies were obtained from the vastus lateralis muscle before and after each exercise bout. Exercise increased muscle aPKC activity at 35%, whereupon no further increase was observed at higher exercise intensities. Activation of aPKC was not accompanied by increased phosphorylation of aPKC Thr410/403. ERK1/2 activity increased in a similar pattern to aPKC, reaching maximal activity at 35%, whereas ERK1 Thr202/Tyr204 and ERK2 Thr183/Tyr185 phosphorylation increased with increasing exercise intensity. Thus, aPKC and ERK1/2 activity in muscle during exercise did not correspond to phosphorylation of sites on aPKC or ERK1/2, respectively, which are considered important for their activation. It is concluded that assessment of aPKC and ERK1/2 activity in muscle using phosphospecific antibodies did not reflect direct activity measurements on immunoprecipitated enzyme in vitro. Thus, estimation of enzyme activity during exercise by use of phosphospecific antibodies should not be performed uncritically. In addition, increase in muscle activity of aPKC or ERK1/2 during exercise is not closely related to energy demands of the muscle but may serve other regulatory or permissive functions in muscle.

Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca2+ release

Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca2+ as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca2+ release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca2+ centric paradigm.

Exercise improves phosphatidylinositol-3,4,5-trisphosphate responsiveness of atypical protein kinase C and interacts with insulin signalling to peptide elongation in human skeletal muscle

We investigated if acute endurance‐type exercise interacts with insulin‐stimulated activation of atypical protein kinase C (aPKC) and insulin signalling to peptide chain elongation in human skeletal muscle. Four hours after acute one‐legged exercise, insulin‐induced glucose uptake was ∼80% higher (N= 12, P < 0.05) in previously exercised muscle, measured during a euglycaemic–hyperinsulinaemic clamp (100 μU ml−1). Insulin increased (P < 0.05) both insulin receptor substrate (IRS)‐1 and IRS‐2 associated phosphatidylinositol (PI)‐3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser473 and Thr308 in skeletal muscle. Interestingly, in response to prior exercise IRS‐2‐associated PI‐3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. This coincided with correspondingly altered phosphorylation of the extracellular‐regulated protein kinase 1/2 (ERK 1/2), p70S6 kinase (P70S6K), eukaryotic elongation factor 2 (eEF2) kinase and eEF2. aPKC was similarly activated by insulin in rested and exercised muscle, without detectable changes in aPKC Thr410 phosphorylation. However, when adding phosphatidylinositol‐3,4,5‐triphosphate (PIP3), the signalling product of PI‐3 kinase, to basal muscle homogenates, aPKC was more potently activated (P= 0.01) in previously exercised muscle. Collectively, this study shows that endurance‐type exercise interacts with insulin signalling to peptide chain elongation. Although protein turnover was not evaluated, this suggests that capacity for protein synthesis after acute endurance‐type exercise may be improved. Furthermore, endurance exercise increased the responsiveness of aPKC to PIP3 providing a possible link to improved insulin‐stimulated glucose uptake after exercise.