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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...

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