Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.

The incidences of obesity and type 2 diabetes are reaching epidemic proportions worldwide. A cardinal feature of these conditions is resistance to the effects of the hormone insulin and a resulting hepatic overproduction of glucose. Insulin resistance is also implicated in a range of liver diseases including non-alcoholic fatty liver disease (NAFLD) and hepatitis C infection. Insulin is released after a meal and acts on liver, skeletal muscle and adipose tissue to reduce blood glucose concentration. In the liver, insulin inhibits the production and release of glucose into the circulation and stimulates its storage as glycogen. Glucagon, on the other hand, is present in the fasting state and causes breakdown of hepatic glycogen along with production of new glucose. This glucose is released from hepatocytes into the circulation. For the studies in this thesis, functional assays to measure various aspects of hepatic glucose metabolism in vitro were developed. This included measuring glucose output into culture medium, hepatocyte uptake of radiolabelled glucose and incorporation into glycogen, and total cellular glycogen content. These assays were used to investigate glucose metabolism in primary rat hepatocytes and FaO rat hepatoma cells. Both cell types responded to physiological concentrations of insulin, showing decreased glucose output and increased glycogen synthesis. Glucagon increased glucose output and reduced glycogen synthesis in primary cells but had no effect on FaO cells. Factors that have been identified that may inhibit or potentiate insulin action were investigated. Increased body iron stores have been linked with insulin resistance. De-ironing patients improves insulin sensitivity, suggesting a causal relationship between iron and insulin resistance. Hepatocytes store the majority of the body’s excess iron. This project investigated the effects of increasing hepatocyte iron stores, through addition of ferric ammonium citrate (FAC), or depleting iron stores by chelation with dipyridyl. Small increases or decreases of iron in primary cells had negative effects on cell viability, resulting in significantly reduced glucose output and glycogen synthesis. Dipyridyl treatment had similar effects on FaO cells as on primary cells but FAC treatment increased FaO glucose output, although significant iron loading was not achieved. With concentrations of FAC and dipyridyl low enough to not significantly influence cell viability, insulin sensitivity was not affected. Adiponectin is an insulin sensitiser and appears to exert this effect primarily through the liver. Adiponectin can also reduce hepatic glucose output (HGO) independent of insulin. It is believed adiponectin mediates its effects in liver, skeletal muscle and adipose tissue through activation of AMP-activated protein kinase (AMPK). In muscle, p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a downstream component of adiponectin signalling. In this study, recombinant human adiponectin was produced and collected in culture medium which was then concentrated. Despite the presence of both high molecular weight (HMW) and low molecular weight (LMW) adiponectin multimers, the concentrated medium had no effect on HGO in the presence or absence of insulin. Concentrated adiponectin medium did not affect AMPK or p38 MAPK phosphorylation in hepatocytes or other cell types previously shown to respond to adiponectin. However, commercially-sourced purified recombinant adiponectin also failed to elicit any observable responses. AICAR and metformin are pharmacological activators of AMPK and were used to treat primary rat hepatocytes and FaO cells. These treatments reduced HGO independent of insulin in both cell types. In primary cells, these reductions were partially inhibited with Compound C, an AMPK inhibitor, suggesting that both AICAR and metformin action is at least partly AMPK dependent. In FaO cells, Compound C only inhibited the AICAR-mediated reduction of glucose output, indicating that metformin may act independently of AMPK in these cells. Compound C significantly inhibited AICAR and metformin-mediated increases in AMPK phosphorylation in primary hepatocytes and FaO cells. There was a trend towards inhibition of AICAR-mediated p38 MAPK phosphorylation with Compound C treatment, suggesting that p38 MAPK may lie downstream of AMPK in hepatocytes. Adenoviral expression of constitutively active (CA) and dominant negative (DN) AMPK in primary rat hepatocytes was used to further study the role of AMPK in hepatic glucose metabolism. Despite significant expression of CA AMPK, phosphorylation of downstream acetyl-CoA carboxylase (ACC) was not affected nor was HGO. CA AMPK did, however, increase phosphorylation of p38 MAPK. DN AMPK completely inhibited AICAR-mediated AMPK phosphorylation and partially inhibited phosphorylation of ACC. In addition, AICAR-mediated phosphorylation of p38 MAPK was inhibited by DN AMPK. Taken together, these results suggest that p38 MAPK is downstream of AMPK in hepatocytes. The implication that p38 MAPK is involved in hepatic AMPK signalling is a novel finding. A greater understanding of this pathway in the liver may identify novel therapeutic targets, leading to improved treatment strategies for metabolic disorders linked to obesity and type 2 diabetes.

Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.

Abstract Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of energy metabolism and mitochondrial biology in high-energy cell types and tissues. The regulation of PGC-1α is versatile, and both transcriptional and post-transcriptional mechanisms play major roles. External stimuli affect PGC-1α-regulation which in turn adapts cellular signals to meet them. For example, conditions like fasting and diabetes mellitus (DM) are known to activate PGC-1α expression in the liver, resulting in enhanced de novo glucose production, gluconeogenesis. In the present study, the mechanisms of hepatic PGC-1α regulation and PGC-1α-regulated functions were elucidated. We found that PGC-1α was induced by oral type 2 diabetes therapeutic metformin, via AMPK and SIRT1, regulating the mitochondrial gene response, against previous assumptions. Simultaneously, gluconeogenesis was repressed by other means. Furthermore, PGC-1α upregulated the anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). PGC-1α also diminished interleukin 1β-mediated inflammatory response in hepatocytes. Novel, xenobiotic and endobiotic metabolizing Cytochrome P450 enzymes regulated by PGC-1α were also identified in this thesis. CYP2A5 was induced by PGC-1α through hepatocyte nuclear factor 4α (HNF-4α) coactivation. Also, vitamin D metabolizing CYP2R1 and CYP24A1 were identified as novel genes regulated by PGC-1α, suggesting a role for PGC-1α in the regulation of active vitamin D levels. The findings presented in this thesis provide insight into the pathology of glucose perturbations such as type 2 diabetes, and stimulate discovery of therapeutic agents to treat this disease. Furthermore, the findings suggest that vitamin D metabolism and energy metabolism are tightly linked, with PGC-1α emerging as a novel mediator. / Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori γ:n koaktivaattori 1α (PGC-1α) on merkittävä glukoosiaineenvaihdunnan ja mitokondrioiden toiminnan säätelijä korkeaenergisissä soluissa ja kudoksissa. PGC-1α:a säädellään monin tavoin: sekä transkriptionaalisella säätelyllä että transkription jälkeisellä muokkauksella on merkittävä rooli. Monet ulkoiset tekijät säätelevät PGC-1α:n aktiivisuutta, joka puolestaan säätelee solunsisäisiä signaalireittejä vastaamaan tähän signaaliin. Esimerkiksi paasto ja diabetes mellitus (DM) ovat fysiologisia tiloja, jotka lisäävät voimakkaasti PGC-1α:n ilmentymistä maksassa, jolloin glukoosin uudistuotanto eli glukoneogeneesi kiihtyy. Tässä väitöskirjassa tutkittiin PGC-1α:n säätelyä sekä PGC-1α -säädeltyjä signaalireittejä maksassa. Osoitimme, että tyypin 2 diabeteslääke metformiini indusoi PGC-1α:n ilmentymistä maksassa, vastoin aikaisempia käsityksiä. PGC-1α indusoitui AMPK:n ja SIRT1:n välityksellä, säädelleen edelleen mitokondriaalisten geenien aktiivisuutta. Samalla glukoneogeneesi kuitenkin repressoitui muilla mekanismeilla. Lisäksi osoitimme, että PGC-1α indusoi tulehdusreaktiota vaimentavaa interleukiini 1 reseptorin antagonistia (IL1Rn). PGC-1α esti interleukiini 1β:n aiheuttamaa tulehdusvastetta hepatosyyteissä. Lisäksi väitöskirjassa tunnistettiin uusia, PGC-1α -säädeltyjä lääkeaineita ja elimistön sisäisiä yhdisteitä metaboloivia sytokromi P450 -entsyymejä (CYP). Hiiren CYP2A5:n ilmentymisen osoitettiin olevan PGC-1α- ja HNF4α-välitteistä. Lisäksi osoitettiin, että D-vitamiinia metaboloivat CYP2R1 ja CYP24A1 ovat uusia PGC-1α -säädeltyjä geenejä. Tämä löydös viittaa siihen, että PGC-1α:lla on rooli aktiivisen D-vitamiinin säätelyssä. Tämän väitöskirjan löydökset lisäävät tietoa glukoosiaineenvaihdunnan häiriöiden kuten tyypin 2 diabeteksen molekulaarisista mekanismeista, joita voidaan hyödyntää mahdollisten uusien lääkeaineiden kehittämisessä. Lisäksi väitöskirjassa osoitettiin, että D-vitamiinimetabolia on kytköksissä energia-aineenvaihduntaan ja että PGC-1α:lla on tässä rooli, jota ei aiemmin ole tunnettu.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21927号 / 生博第412号 / 新制||生||54(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 影山 龍一郎, 教授 垣塚 彰 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hep- atocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKa activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 signifi- cantly activated AMPKa and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a po- tential treatment option for HCC.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated when cellular energy is low and causes muscle and other cells to increase glucose uptake and fat oxidation, diminish lipid synthesis, and alter expression of various genes. AMPK activity is diminished in animals with the metabolic syndrome, though the mechanisms causing this reduction are unknown. To examine nutrient-induced changes in AMPK activity over time and factors that may regulate it, we compared rat muscle incubated with high glucose (HG) (30min-2h) and muscle of glucose-infused rats (3-8h) with appropriate controls. In addition to diminished AMPK activity (measured by the SAMS peptide assay) and phosphorylation of its activation loop at Thr172, we observed increased muscle glycogen, phosphorylation of AMPK’s α1/α2 subunit at Ser485/491, and PP2A activity, and decreased SIRT1 expression, all of which have been shown to diminish AMPK activity. Dysregulation of one or more of these factors could contribute to pathophysiological changes leading to metabolic syndrome associated disorders. Since recent studies suggest phosphorylation at Ser485/491 may play an important role in AMPK inhibition, we sought to determine how phosphorylation of this site is regulated. We investigated whether insulin or diacylglycerol (DAG) signaling pathways may be involved, since both are increased in at least one of the HG models. Akt and Protein Kinase (PK)D1 phosphorylated AMPK at Ser485/491 and diminished its activity in C2C12 myotubes, downstream of insulin and the DAG-mimetic PMA, respectively. Additionally, p-AMPK Ser485/491 was increased in muscle and liver of fed versus fasted mice and liver of diabetic mice. Our results suggest that Akt- and PKD1-mediated inhibition of AMPK via Ser485/491 phosphorylation may inhibit energy-metabolizing processes, while favoring energy-storing processes. Our results highlight the fact that phosphorylation of Ser485/491 can inhibit AMPK activity independent of changes in p-AMPK Thr172, a measure which is often used as a readout of AMPK activity. We hypothesize that Akt-mediated inhibition of AMPK is an acute, physiological response to insulin, whereas PKD1-mediated inhibition may be associated with more chronic pathophysiological changes. Thus, PKD1 inhibition or prevention of Ser485/491 phosphorylation may represent new strategies for therapeutic AMPK activation as treatment for the metabolic syndrome.

<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.