Recent research suggests that LKB1 is the major AMP-activated protein kinase kinase (AMPKK). Peroxisome-proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣) is a master coordinator of mitochondrial biogenesis. Previously we reported that skeletal muscle LKB1 protein increases with endurance training. The purpose of this study was to determine whether training-induced increases in skeletal muscle LKB1 and PGC-1␣ protein exhibit a time course and intensity-dependent response similar to that of citrate synthase. Male Sprague-Dawley rats completed endurance-and interval-training protocols. For endurance training, rats trained for 4, 11, 25, or 53 days. Interval-training rats trained identically to endurance-trained rats, except that after 25 days interval training was combined with endurance training. Time course data were collected from endurance-trained red quadriceps (RQ) after each time point. Interval training data were collected from soleus, RQ, and white quadriceps (WQ) muscle after 53 days only. Mouse protein 25 (MO25) and PGC-1␣ protein increased significantly after 4 days. Increased citrate synthase activity, increased LKB1 protein, and decreased AMPKK activity were found after 11 days. Maximal increases occurred after 4 days for hexokinase II, 25 days for MO25, and 53 days for citrate synthase, LKB1, and PGC-1␣. In WQ, but not RQ or soleus, interval training had an additive effect to endurance training and induced significant increases in all proteins measured. These results demonstrate that LKB1 and PGC-1␣ protein abundances increase with endurance and interval training similarly to citrate synthase. The increase in LKB1 and PGC-1␣ with endurance and interval training may function to maintain the traininginduced increases in mitochondrial mass. adenosine 5Ј-monophosphate-activated protein kinase, AMP-activated protein kinase kinase; diabetes; MO25, Ste-20-related adaptor protein THE INCIDENCE OF TYPE 2 DIABETES and obesity is increasing at an alarming rate. As of 2002, 8.7% of Americans over age 20 yr and 18.3% of Americans over age 60 yr were diabetic (1). Type 2 diabetes is strongly associated with obesity and is characterized primarily by a reduction in insulin-stimulated glucose uptake. Type 2 diabetes is often accompanied by a decreased skeletal muscle mitochondrial content and a higher-than-normal proportion of type IIx (IIb) muscle fibers (9,24,31,37,48). Regular exercise prevents type 2 diabetes directly by increasing insulin sensitivity and indirectly by decreasing adiposity. Physical activity also increases skeletal muscle mitochondrial mass and induces an increased expression of type IIa myosin heavy chain (MHC) and a decreased expression of type IIx (IIb) MHC (40). Training intensity regulates fiber typespecific training adaptations (2, 57). Higher-intensity training is required for the maximal recruitment and training of type IIx (IIb) fibers (16,46). Hence, interval training might be particularly important for type 2 diabetics.Research during the past decade has characterized some of t...

Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.

. Endurance training increases LKB1 and MO25 protein but not AMP-activated protein kinase kinase activity in skeletal muscle. Am J Physiol Endocrinol Metab 287: E1082-E1089, 2004. First published August 3, 2004 doi:10.1152/ajpendo.00179.2004 and STRAD has been identified as an AMP-activated protein kinase kinase (AMPKK). We measured relative LKB1 protein abundance and AMPKK activity in liver (LV), heart (HT), soleus (SO), red quadriceps (RQ), and white quadriceps (WQ) from sedentary and endurance-trained rats. We examined trained RQ for altered levels of MO25 protein and LKB1, STRAD, and MO25 mRNA. LKB1 protein levels normalized to HT (1 Ϯ 0.03) were LV (0.50 Ϯ 0.03), SO (0.28 Ϯ 0.02), RQ (0.32 Ϯ 0.01), and WQ (0.12 Ϯ 0.03). AMPKK activities in nanomoles per gram per minute were HT (79 Ϯ 6), LV (220 Ϯ 9), SO (22 Ϯ 2), RQ (29 Ϯ 2), and WQ (42 Ϯ 4). Training increased LKB1 protein in SO, RQ, and WQ (P Ͻ 0.05). LKB1 protein levels after training (%controls) were SO (158 Ϯ 17), RQ (316 Ϯ 17), WQ (191 Ϯ 27), HT (106 Ϯ 2), and LV (104 Ϯ 7). MO25 protein after training (%controls) was 595 Ϯ 71. Training did not affect AMPKK activity. MO25 but not LKB1 or STRAD mRNA increased with training (P Ͻ 0.05). Trained values (%controls) were MO25 (164 Ϯ 22), LKB1 (120 Ϯ 16), and STRAD (112 Ϯ 17). LKB1 protein content strongly correlated (r ϭ 0.93) with citrate synthase activity in skeletal muscle (P Ͻ 0.05). In conclusion, endurance training markedly increased skeletal muscle LKB1 and MO25 protein without increasing AMPKK activity. LKB1 may be playing multiple roles in skeletal muscle adaptation to endurance training. adenosine 5Ј-monophosphate-activated protein kinase; diabetes; serine-threonine kinase-11; Ste20-related adaptor protein LKB1 (SERINE-THREONINE STK11) in complex with the regulatory proteins MO25 and Ste20-related adaptor protein (STRAD) has recently been identified as a major upstream kinase for the AMP-activated protein kinase (AMPK) (16,42,49). LKB1 requires association with STRAD for catalytic activity that is enhanced by binding to MO25 (1, 16). MO25 stabilizes the interaction between LKB1 and STRAD and is thought to act as a scaffolding protein (5, 30). AMPK is a master metabolic regulator responsible for modulating cellular responses to an energy challenge (15,38,47). These responses include increased glucose uptake (3,18,22,29), increased fatty acid oxidation (29, 46), decreased protein synthesis (4,9,20), and induction of mitochondrial biogenesis (2, 48). AMPK has been the subject of intense investigation because of its potential as a therapeutic target for antidiabetic drugs (25,32,37). Full activation of AMPK requires phosphorylation of its activation loop at Thr 172 by an AMPK kinase (AMPKK) (17, 44). Thus the LKB1-STRAD-MO25 AMPKK complex may be a key regulator of the downstream effects of AMPK, including the regulation of exercise-induced glucose uptake. LKB1 is a tumor suppressor protein regulating cell proliferation and polarity (7). Inactivating mutations in LKB1 were discovered as the causative...

(LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant ␣1␤1␥1 or ␣2␤2␥2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the ␣1 or ␣2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction. adenosine monophosphate-activated protein kinase kinase; adenosine triphosphate; exercise; metabolism; skeletal muscle MUSCLE CONTRACTION RESULTS IN ACTIVATION of the AMP-activated protein kinase (AMPK) (36). Acute activation of AMPK initiates changes in cellular metabolism, enabling working skeletal muscle to better meet an energy challenge (7,8,35). These metabolic responses include increased glucose transport (11,14,19), increased fatty acid oxidation (19, 32), and decreased protein synthesis (3,12,24). Chronic activation of AMPK also results in adaptive transcriptional events (16), including mitochondrial biogenesis (2, 37, 40), which make skeletal muscle more robust when encountering subsequent energy challenges.AMPK requires phosphorylation on its activation loop at Thr 172 by an AMPK kinase (AMPKK) for activation (10,29,34). The tumor suppressor kinase LKB1 (STK-11) is a major AMPKK (9,28,38). Recently, generation of a skeletal musclespecific LKB1 knockout mouse demonstrated that LKB1 is the major AMPKK in skeletal muscle (26). LKB1 requires association with the regulatory proteins Ste20-related adapter protein (STRAD) and mouse protein 25 (MO25) for full activity (4,5,9). An increase in the intracellular AMP-to-ATP ratio induces a conformational change in AMPK by interacting with 4 cystathione ␤-synthase (CBS) domains on the ␥-subunit (1, 27). This conformational change makes AMPK a suitable substrate for the constitutively active LKB1/STRAD/MO25 complex (LKB1). LKB1 activity is not increased by muscle contraction (25). AMPK activity has also been reported to be regulated by creatine-to-creatine phosphate and NAD-to-NADH ratios (22,23).Muscle contraction results in depletion of creatine phosphat...