Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase, AMPKK. The LKB1-STRAD-MO25 complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study we tested an array of metabolites including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose 1,6-bisphosphate (F1,6-P2), 3-phosphoglycerate (3PG), glucose-1-phosphate (G1P), glucose-1,6-bisphosphate (G1,6-P2), adenosine diphosphate (ADP), carnitine (Carn), acetyl-carnitine (Acarn), inosine monophosphate (IMP), inosine, and ammonia for allosteric regulation. We found that 3PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.

The LKB1/STRAD/MO25 complex (LSMK) has been identified as the major upstream kinase for AMP-activated protein kinase (AMPK). PKA phosphorylates LKB1 at the Ser428 residue in humans and Ser431 residue in mice. We investigated PKA as an upstream kinase for LSMK. LKB1 that had been incubated with PKA prior to incubation with AMPK experienced up to a 51% increase in AMPK Kinase activity compared to LKB1 alone (p < 0.05). When blocked with a PKA Inhibitor, the kinase effect of PKA on LKB1 was eliminated. Rat epitrochlearis muscle tissue incubated with epinephrine experienced no increase in AMPK activity compared with controls indicating that epinephrine does not cause AMPK activity in this type of tissue. In conclusion, phosphorylation by PKA can increase the AMPKK activity of LKB1-STRAD-MO25 in vitro. Because LKB1 has been found to be constitutively active, it is postulated that phosphorylation by PKA may act to enhance LKB1-AMPK interaction and thus achieve its effect.

Activation of AMP-activated protein kinase (AMPK) results in the upregulation of several intracellular systems which help to prepare a cell for a high energy challenge. The magnitude of the AMPK response to a 10 min bout of exercise has been found to decrease in red quadriceps (RQ) following training, while putative AMPK roles seem to be maintained; specifically, the biogenesis of mitochondria and higher levels of hexokinase II and glucose transporter 4 (GLUT4). If the AMPK response to exercise is responsible in part for these adaptations, how can they be maintained if the AMPK response is attenuated? The purpose of this study was to determine whether phosphorylation of AMPK in RQ increases during 2-hr training bouts after rats have trained for 8 wks. Male Sprague-Dawley rats ran up to 30 m/min up a 15% grade, 2 hr/day for 8 wks. On the final bout of exercise, trained rats ran for 0 (TRC), 30 (TR1), or 120 min (TR2) up a 15% grade at 30 m/min. Red quadriceps (RQ), soleus, and white quadriceps (WQ) were immediately collected and frozen for analysis. Citrate synthase activity increased in RQ (79 ± 3 vs. 37 ± 4 µmol/g/min) and soleus (64 ± 4 vs. 35 ± 2 µmol/g/min) but not in WQ compared to non-trained controls. In trained rats, maximal increases in T-172 phosphorylation of AMPK occurred after 30 min of exercise (relative values = 1.29 ± 0.06 vs. 1.00 ± 0.06). AMPK phosphorylation did not change significantly in trained rats that ran for 2 hrs (1.31 ± 0.09) compared to rats that ran for 30 min. Similarly, maximal increases in AMPK activity in trained rats occurred after 30 min of exercise (pmoles/min/mg = 2.67 ± .05 vs. 1.09 ± .41) and AMPK activity did not change significantly in trained rats that ran for 2 hrs (2.79 ± .17) compared to rats that ran for 30 min. Previous studies demonstrated a 2−3 fold increase in AMPK activity in non-trained rats after 30 min of exercise at lower work rates. These results demonstrate that the AMPK response to exercise is attenuated even after two-hr bouts of exercise. This implies that the increase in mitochondrial oxidative enzymes, GLUT4, and hexokinase II may be maintained by signals other than the AMPK signaling system. The CREB signaling pathway is one such system. Western analysis of phospho-CREB (Ser133) showed a statistically significant increase in phospho-CREB content in trained rats relative to control. No change in phospho-CREB protein expression was observed between TRC, TR1, and TR2 rats. Significant increases of muscle phospho-CREB content in TRC relative to untrained rats suggest that CREB remains phosphorylated in trained rats even after 24 hrs of rest. Accordingly, chronically increased phospho-CREB in muscle of trained rats relative to controls may explain in part how increased levels of mitochondria are maintained in the face of reduced AMPK response. Alternatively, the attenuated AMPK response may still be above the threshold required for inducing adaptations to endurance training.

High fat feeding increases hepatic fat accumulation and is associated with hepatic insulin resistance. AMP Activated Protein Kinase (AMPK) is thought to inhibit lipid synthesis by the acute inhibition of glycerol-3-phosphate acyltransferase (GPAT) activity and transcriptional regulation via SREBP-1c. The purpose of this study was to determine if chronic activation of AMPK prevented an increase in GPAT1 activity in rats fed a high fat diet. Rats were fed a control (C), or a high fat (HF) diet (60% fat) for 6 weeks and injected with saline or a daily AICAR dose of 0.5 mg/g body weight. Chronic AMPK activation by AICAR injections resulted in a significant reduction in hepatic triglyceride accumulation in both the C and HF fed animals (C, 5.5±0.7; C+AICAR, 2.7 ±0.3; HF, 21.8±3.3; and HF+AICAR, 8.0±1.8 mg/g liver). HF feeding caused an increase in total GPAT and GPAT1 activity which was not affected by chronic AMPK activation (GPAT1 activity vs. C, C+AICAR, 92±19%; HF, 186±43%; HF+AICAR, 234±62%). Markers of oxidative capacity, including citrate synthase activity and cytochrome c abundance, were not affected by chronic AICAR treatment. Interestingly, HF feeding caused a significant increase in LCAD (up 66% from C), a marker of fatty acid oxidation capacity. These results suggest that chronic AMPK activation limits hepatic triglyceride accumulation independent of a reduction in total GPAT1 activity.

Skeletal muscle has the ability to increase in size (hypertrophy) after resistance is placed upon it. This hypertrophy is marked by significant upregulation of the mammalian target of rapamycin (mTOR) and its downstream targets. The upstream kinases, protein kinase B (also known as Akt) and AMP-activated protein kinase (AMPK), are two of the many regulators of the mTOR pathway. Recent studies suggest that the widely consumed neuroactive compound caffeine could potentially inhibit mTOR by acting through Akt and/or AMPK. The purpose of this thesis was to: 1) determine if caffeine can inhibit the mTOR pathway and ultimately attenuate skeletal muscle hypertrophy and 2) determine if this inhibition is through LKB1, an upstream regulator of AMPK. First, 3 month old male rats underwent unilateral tenotomy of the gastrocnemius, resulting in overloading (OVLD) of the synergistic plantaris muscle. The contralateral limb was sham-operated (SHAM) on. Rats were given ad libitum access to tap water or tap water + caffeine (1 g/L). The OVLD procedure resulted in significant hypertrophy of the plantaris which was attenuated after 1 wk of caffeine treatment. However, after two wks this effect was not observed. mTOR targets were examined in both the SHAM and OVLD plantaris muscle which showed significant upregulation with OVLD but no impact with caffeine treatment. Akt and AMPK was also assessed in the plantaris muscle which showed diminished Akt phosphorylation in 1 wk treated rats while the phosphorylation of AMPK remained relatively unaffected. Notably, caffeine caused decreased atrophy of the tenotomized gastrocnemius after 1 wk along with decreased body weight gains, food consumption, and retroperitoneal fat pad weight in both 1 and 2 wk treated rats. Second, to elucidate how caffeine could be impacting the mTOR pathway and how LKB1/AMPK might be involved, skeletal muscle specific LKB1 knockout (skmLKB1-KO) mice were subjected to high-frequency electrical stimulation (HFES) of the sciatic nerve resulting in contraction of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles against the larger gastrocnemius. All mice were given an intraperitoneal injection of saline or saline + caffeine (20 mg/kg BW at 1 g/L). HFES resulted in marked upregulation of mTOR targets in the TA/EDL of mice 0, 3, and 8 h post HFES. mTOR targets remained relatively unchanged with caffeine treatment. We also observed that these markers were consistently upregulated in our skmLKB1-KO mice with or without HFES. Our findings indicate that caffeine, at physiological concentrations, does not impact anabolic signaling. Furthermore, diminished LKB1 levels resulted in increased levels and activation of markers of protein synthesis.

AMPK is a master regulator of cellular energy homeostasis. The gain-of-function AMPKg3 R225W mutation in human skeletal muscle increases resistance to fatigue during exercise, mitochondrial content, and glycogen storage. We demonstrate that primary myotubes exhibit increased OCR, decreased ECAR, increased FAO, and increased activities of several mitochondrial complexes. To examine whether functional effects are attributable to mitochondrial content, we inhibited AMPK; differences between R225W and control were diminished. Glycogen phosphorylase inhibition demonstrated normal respiration independent of glycogen. We examined markers of quality/quantity control of mitochondria. In R225W muscle, fusion markers increased, biogenesis markers remained unchanged, mTOR pathway was inhibited, and there was greater capacity for autophagic flux and mitophagy. We thus determine that bioenergetic effects of R225W are in part due to active AMPK, but also due to capacity for more robust mitochondria. Overall, R225W provides a model for evaluating effects of AMPK, and new avenues toward treatment of metabolic disease.

The neuromuscular junction (NMJ) exhibits an extraordinary capacity for adaptation and plasticity throughout an individual's lifespan. This remarkable adaptability assumes a central role in safeguarding optimal neuromuscular function and counteracting neurodegenerative processes commonly associated with aging and prevalent neuromuscular disorders. The plasticity of the NMJ is under the influence of its cellular constituents, including the ⍺-motoneuron and the innervated muscle fiber. Among the diverse array of regulatory molecules, AMP-activated protein kinase (AMPK) plays a pivotal role in governing the phenotype of these cellular components, thereby potentially contributing to synaptic modifications. To explore the regulatory role of AMPK on the NMJ phenotype, we undertook a comprehensive investigation encompassing transgenic, pharmacologic, and physiologic manipulations of this kinase. In Study 1, we investigated the significance of skeletal muscle AMPK during aging, revealing its necessity in preserving NMJ integrity. Moreover, we observed that pharmacological and physiological activation of AMPK result in an enhanced synaptic gene profile in young animals, suggesting its role in NMJ modulation. Building upon these insights, we validate the stimulatory effects of a pan-AMPK activator, MK-8722 (MK), in the context of a prevalent neuromuscular disorder, Duchenne Muscular Dystrophy (DMD). Our investigations demonstrated that MK effectively evoked AMPK activation and downstream signaling in dystrophic muscle, providing the experimental foundations our third study. Here, we assess of the chronic effects of daily MK treatment in a pre-clinical DMD model and revealed significant improvements in mitochondrial health, neuromuscular function, and a reduction in muscle fibrosis and fatigue. Taken together, these findings support a critical role of AMPK in neuromuscular plasticity and highlight the kinase as a promising therapeutic target for muscular dystrophy. / Dissertation / Doctor of Philosophy (PhD) / The neuromuscular junction (NMJ) plays a vital role in maintaining muscle function and countering aging and neuromuscular disorders. This thesis investigated the role of AMP-activated protein kinase (AMPK) in neuromuscular biology during conditions of health and disease. We conducted various experiments involving genetic modifications, drug treatments, and exercise. First, we determined that AMPK is necessary to maintain the NMJ during aging. Stimulation of AMPK with a potent activator, MK-8722 (MK), led to elevated NMJ-related gene expression. We then shifted our focus to the most prevalent neuromuscular disorder, Duchenne Muscular Dystrophy (DMD). Our results showed that MK activated AMPK in dystrophic mice, prompting us to further investigate the long-term effects of daily treatment in a pre-clinical DMD model. Repeated MK treatment significantly improved neuromuscular function and reduced the symptoms of DMD. Together, our comprehensive investigation demonstrates the critical role of AMPK in shaping neuromuscular plasticity during healthy and diseased conditions.

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