<p>In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration.</p><p>Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.</p>

Muscle fatigue is an intrinsic myoprotective process that prevents damaging ATP depletion during intense or prolonged exercise by limiting ATP demand when ATP production becomes insufficient. One mechanism of fatigue involves a reduction in membrane excitability with the opening of ATP-sensitive K+ (KATP) and ClC-1 Cl- channels, resulting in submaximal sarcoplasmic reticulum Ca2+ release and reduced force generation, but the intracellular signalling pathways for this process is unknown. As a first step toward understanding this process, the objective of this study was to test the hypothesis that adenosine receptors (ARs) and AMPK trigger fatigue when a metabolic stress occurs during muscular activity. Compared to control conditions, a pan-activation of ARs with 10 µM adenosine and NECA initially reduced the fatigue rate during the first 60 s of a 3 min fatigue bout triggered with 1 tetanic contraction every s. An activation of the A1 adenosine receptor (A1R) with 10 and 20 µM ENBA resulted in faster rate of fatigue; an effect blocked by 5 µM DPCPX, an A1R antagonist. At 10 and 20 µM, adenosine, NECA, and ENBA activated AMPK via an increased in T172 phosphorylation. At 10 µM, MK8722, an AMPK agonist, initially caused a reduction in fatigue rate during the first 60 s followed by an increased fatigue rate during the last 2 min of the fatigue bout. Co-activation of ARs and AMPK did not give rise to either an additive or synergistic effect. FDB from AMPK α1-/- and α2-/- mice had faster fatigue rate and greater increased in unstimulated force compared to FDB from AMPK α1+/+ and α2+/+ mice. It is suggested that ARs and AMPK play a role in the mechanism of fatigue when a metabolic stress develops during muscle activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) and AMP-activated protein kinase (AMPK) in muscles from control (C) and endurance trained (T) rats. Rats were trained 5 days/wk, 2 hr/d for 8 wks at a final intensity of 32 m/min up a 15% grade with 30 second sprints at 52 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in T and C rats for 5 min at frequencies of 0.4/sec and 1/sec. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately 2-fold in response to training. Phosphorylation of AMPK determined by western blot was increased at both stimulation rates in both control and trained rats. AMPK activity of both the α1 and α2 isoforms (immunoprecipitates) also increased at both stimulation rates in both C and T rats. AMPKK activity was strikingly lower in both resuspended polyethylene glycol (PEG) precipitates and 1200 x g supernatant of the crude homogenate of muscle extracts from the trained compared to control rats. AMPKK activity did not increase in either T or C in response to electrical stimulation even though phospho-AMPK did increase. Interestingly, AMPKK activity in the 1200 x g supernatant of the crude homogenate actually decreased upon stimulation in the control rats. These results suggest that AMPKK is activated during electrical stimulation by mechanisms other than covalent modification. Possibilities include AMP-induced optimization of the phosphorylation site on the target protein, contraction-induced changes in undefined allosteric modulators, and contraction-induced association with other proteins. (Study approved by the IACUC and supported by NIH RO1 AR41438.)

AMP-activated protein kinase (AMPK), a heterotrimeric protein which serves as a metabolic master switch in skeletal muscle, is a research target for the pharmaceutical treatment and prevention of type 2 diabetes. The expression of all of the isoforms of the subunits of AMPK and AMPK activity are increased in skeletal muscle tissue of hyperthyroid rats. Activity of AMPK is regulated by an upstream kinase (AMPKK). The LKB1-STRAD-MO25 complex is a major AMPKK in skeletal muscle. This experiment was designed to determine whether the increase in AMPK activity is accompanied by a thyroid hormone-induced increase in the expression of the LKB1-STRAD-MO25 complex. LKB1-STRAD-MO25 complex protein expression was determined by Western blots in control rats, in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 4 weeks, and in rats given 0.01% propylthiouracil (PTU) in drinking water for 4 weeks. The relative expression of LKB1, MO25, and STRAD, as well as PGC-1α, increased in the soleus of thyroid hormone treated rats vs. the controls. MO25 mRNA increased with thyroid hormone treatment, and STRAD mRNA increased with PTU treatment. Phospho-AMPK and phospho-ACC increased in response to electrical stimulation in muscles of all treatment groups, but was most markedly increased in hyperthyroid rats. Thyroid hormone treatment also increased the amount of phospho-CREB in the soleus, heart, and red quadriceps. These data provide evidence that thyroid hormone partially controls expression of the LKB1-STRAD-MO25 complex, as well the subsequent activation of AMPK.

In liver tissue, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of LKB1, MO25α/β, and STRADα/β proteins; however, this complex has yet to be characterized in skeletal muscle. In this report, we demonstrate the expression of the LKB1-MO25-STRAD AMPKK complex in adult skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these complex-forming proteins. To facilitate this characterization we used control (ctrl) and muscle-specific LKB1 knockout (LKB1-/-) mice. LKB1 detection in untreated ctrl and LKB1-/- muscle lysates revealed two protein bands at approximately 50 and 60 kDa; although, only the heavier band was significantly diminished in LKB1-/- samples (ctrl: 55±2.5 AU; LKB1-/-: 13±1.5 AU; p<0.01), suggesting that LKB1 is not represented at 50 kDa as cited previously. Detection of LKB1 at the higher molecular weight was further confirmed following purification of the AMPKK complex using polyethylene glycol (PEG) (ctrl: 43±5 AU; LKB1-/-: 8.4±4 AU; p<0.01). Following ion-exchange-fast protein liquid chromatography (FPLC) the low protein band was undetectable in ctrl and LKB1-/- fractions. Mass spectrometry of PEG-treated ctrl lysates confirmed LKB1 protein detection in the 60 kDa protein band while none was detected in the 50 kDa band. Co-immunoprecipitation assays demonstrated associations between all combinations of LKB1, MO25, and STRAD in LKB1-positive samples, confirming proper complex formation. Quantitative-PCR revealed significantly reduced expression of MO25α and STRADβ in LKB1-/- muscle. Lastly, detection of CaMKKα/β protein in ctrl and LKB1-/- muscle lysates confirmed the presence of another AMPKK in muscle. Interestingly, CaMKKβ protein is increased in LKB1-/- muscle (ctrl: 19±4.3 AU; LKB1-/-: 47±9.2 AU; p<0.05) without an increase in mRNA levels, suggesting compensation for null LKB1 expression. In all, these findings confirm the presence of the LKB1-MO25-STRAD complex in adult skeletal muscle, suggest a novel post-translational modification of LKB1, and identify a potential compensatory mechanism for loss of LKB1 protein in skeletal muscle.

AMP-activated protein kinase (AMPK), a metabolic regulator, acts in opposition to many of the effects of aging and may provide insights into the development of sarcopenia. However, the effect of aging on AMPK activation is unclear. The purpose of this dissertation was to: 1) clarify the controversy concerning the activation of AMPK in response to endurance-like exercise in aged skeletal muscle; 2) address mechanisms for the age-associated alterations in AMPK activation; and 3) address the known benefits of chronic AICAR treatment in aged skeletal muscle. First, to clarify the effect of age on AMPK activation, young adult (YA) (8 mo.) and old (O) (30 mo.) male Fischer344 x Brown Norway F1 hybrid rats received an in situ bout of endurance-type contractions produced via electrical stimulation of the sciatic nerve (STIM). AMPK activation was attenuated in aging muscle as demonstrated by decreased AMPKα phosphorylation and AMPKα2 protein content and activity in O vs. YA muscle after STIM. In contrast, AMPKα1 content was greater in O vs. YA muscle, and α1 activity increased with STIM in O but not YA muscles. Second, the effect of age on the AMPK heterotrimer composition and nuclear localization was assessed as mechanisms for the altered AMPK activation. The AMPK heterotrimer composition was altered in aging skeletal muscle with lower AMPKγ2 and γ3 content and decreased association of AMPKγ3 with AMPKα1 and α2. Furthermore, activation of AMPK is known to increase translocation of AMPK to the nucleus in YA muscle; however, translocation of phosphorylated AMPK, AMPKα2, and AMPKγ3 were impaired in the aging rat muscle after STIM. Finally, chronic activation of AMPK with 5'-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is known to increase mitochondrial content, activate autophagy, and repress protein synthesis; pathways that are altered with aging. The known benefits of chronic AICAR treatment were assessed in YA (5 mo.) and O (23 mo.) male C57Bl/6 mice. Mice were treadmill tested prior to and after one month of AICAR treatment. In vitro muscle contractions were performed following AICAR treatment. AICAR treatment improved the O mice treadmill endurance and the YA mice rate of fatigue and recovery. Additionally, AICAR increased citrate synthase activity, decreased SQSTM1/p62 protein content , and decreased Myf6 protein content in both the YA and O mice suggesting increased mitochondrial activity, autophagy, and decreased muscle regeneration. Therefore, chronic AICAR treatment may alter metabolic pathways to improve the exercise response in both YA and O mice.

No description available.

Brown (BAT) and white (WAT) adipose tissues are significant contributors to whole-body energy homeostasis. A disturbance in their metabolic function could result in the development of obesity and subsequent metabolic complications. The energy-sensing enzyme of the cell, AMP-activated protein kinase (AMPK), has been vastly studied in skeletal muscle and liver, but its role in BAT and WAT metabolism is elusive. We generated an inducible, adipocyte-specific knockout mouse model for the two AMPK β subunits (iβ1β2AKO) and found that iβ1β2AKO mice were intolerant to cold, and resistant to β3-adrenergic activation of BAT and browning of WAT. These defects in BAT activity were not due to the AMPK-ACC axis, but instead were due to compromised integrity of mitochondria. Mitochondrial morphology, function, and autophagy were all distorted in iβ1β2AKO mice, measured via transmission electron microscopy (TEM), respiration, and immunoblotting, respectively. These findings provide strong evidence that adipocyte AMPK regulates a fine-tuned program that responds to environmental and pharmacological inputs by maintaining mitochondrial integrity through autophagy and subsequent mitochondrial biogenesis in chronic settings. / Thesis / Master of Science (MSc) / Traditionally, there are two types of adipose tissue that appear and function differently. White adipose tissue (WAT) has evolved to store away energy in an efficient manner for later use. In contrast, brown adipose tissue (BAT) is a unique organ in mammals that has evolved over time to maintain body temperature. In essence, BAT has the ability to burn away calories as heat and is a promising therapeutic target to combat obesity and metabolic diseases such as type 2 diabetes. In our study, we have identified a potential factor that not only promotes BAT activity, but also promotes WAT to function more like BAT. By targeting this factor through drugs, there is potential to increase resting metabolic rate and fight the global epidemic of obesity.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25024号 / 生博第515号 / 新制||生||69(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 見学 美根子, 教授 上村 匡, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM