Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis

The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1alpha, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIbeta) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS Unhealthy lifestyles predispose to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 minutes/day, 5 days/week during 32 weeks. RESULTS After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3) vs 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the Non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS These data show a benefit of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

The Effect of Heavy Metal Stress on Avian Proximal Tubule Urate Secretion

In both humans and birds, urate is an important antioxidant when maintained at normal plasma concentrations. Though human kidneys primarily reabsorb filtered urate, while those of birds perform mostly secretion, both maintain urate levels at ~300microM. The importance of maintaining urate levels within the homeostatic range was observed when the study of several prominent diseases revealed an association with hyperuricemia. This study examined the effect of elevated zinc concentration on avian urate secretion. Here, acute exposure of chicken proximal tubule epithelial cells (cPTCs) to zinc stress had no effect on urate secretion, but prolonged zinc-induced cellular stress inhibited active transepithelial urate secretion with no change in Mrp4 expression, glucose transport, or transepithelial resistance. Moreover, zinc had no effect on urate transport by isolated brush border membrane vesicles, suggesting involvement of a more complex cellular stress adaptation. Previous work has demonstrated that AMP-activated protein kinase (AMPK), a critical metabolic regulator, conserves energy during cellular stress by shutting down ATP-utilizing processes and activating ATP-generating processes. Pharmacological activation of AMPK by AICAR produced decreased urate secretion by cPTCs similar to the effect seen with prolonged exposure to zinc, while the AMPK inhibitor Compound C prevented both AICAR and zinc inhibition of urate secretion, suggesting a stress induced mechanism of regulation. Supported by NSF. IACUC #A08-046.

Molecular mechanisms for the insulin sensitizing activity of rexinoids in skeletal muscle of diabetic (Db/Db) mice

Rexinoids are synthetic agonists for the retinoid X receptors (RXRs), a member of the nuclear receptor family of ligand-activated transcription factors. Rexinoids have been shown to lower serum glucose and insulin levels in animal models of type 2 diabetes. However the mechanisms that are responsible for the insulin-sensitizing action of rexinoids are largely unknown. Skeletal muscle accounts for the majority of insulin-regulated whole-body glucose disposal and impaired insulin action in muscle is an important contributor to the pathophysiology of type 2 diabetes. Glucose transport is a rate-limiting step in glucose utilization. The goal of these studies is to examine the mechanisms of the anti-diabetic activity of rexinoids in skeletal muscle of diabetic db/db mice. The results we have obtained showed that treatment of db/db mice with rexinoids for two weeks resulted in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Insulin stimulates glucose transport in muscle via the regulation of both the insulin receptor substrate-1 (IRS-1)/Akt pathway and the Cbl-associated protein (CAP)/Cbl pathway. Rexinoids increased the insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation without effects on the activity of the CAP/Cbl pathway. The effects of rexinoids on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Serine 307 phosphorylation as well as qualitative and quantitative alterations in the fatty acyl-CoAs present within the muscle cells. In addition, rexinoids increased the expression of uncoupling protein 3 (UCP3) and activation of AMPK in diabetic muscle. This effect may also enhance the IRS-1/Akt signaling. We believe that it is the concerted activation of the IRS-1/Akt and AMPK signaling systems, a pharmacological mechanism that as far as we know, is unique to rexinoids, that results in the anti-diabetic effects of these drugs. Our results also suggest that the glucose-lowering mechanism of rexinoids is distinct from that of the thiazolidinediones (TZDs), peroxisome proliferator-activated receptor γ (PPARγ) agonists with well-characterized anti-diabetic activity. Rexinoids appear to represent a novel class of insulin sensitizers, with potential applications for the treatment of type 2 diabetes. ^

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

DIRECT EFFECTS OF METFORMIN ON PI3K AND RAS SIGNALING IN ENDOMETRIAL CANCER

Metformin has antiproliferative effects through the activation of AMPK and has gained interest as an antineoplastic agent in several cancer types, although studies in endometrial cancer (EC) are limited. The aims of this project were to evaluate pathways targeted by metformin in EC, investigate mechanisms by which metformin exerts its antiproliferative effects, and explore rational combination therapies with other targeted agents. Three EC cell lines were used to evaluate metformin’s effect on cell proliferation, PI3K and Ras-MAPK signaling, and apoptosis. A xenograft mouse model was also used to evaluate the effects of metformin treatment on in vivo tumor growth. These preliminary studies demonstrated that K-Ras mutant cell lines exhibited a decreased proliferative rate, reduced tumor growth, and increased apoptosis in response to metformin compared to K-Ras wild-type cells. To test the hypothesis that mutant K-Ras may predict response to metformin, murine EC cells with loss of PTEN and expressing mutant K-RasG12D were transfected to re-express PTEN or have K-Ras silenced using siRNA. While PTEN expression did not alter response to metformin, cells in which K-Ras was silenced displayed reduced sensitivity to metformin. Mislocalization of K-Ras to the cytoplasm is associated with decreased signaling and induction of apoptosis. Metformin’s effect on K-Ras localization was analyzed by confocal microscopy in cells expressing oncogenic GFP-K-RasG12V. Metformin demonstrated concentration-dependent mislocalization of K-Ras to the cytoplasm. Mislocalization of K-Ras to the cytoplasm was confirmed in K-Ras mutant EC cells (Hec1A) by cell fractionation in response to metformin 1 and 5 mM (p=0.008 and p=0.004). This effect appears to be AMPK-independent as combined treatment with Compound C, an AMPK inhibitor, did not alter K-Ras localization. Furthermore, treatment of EC cells with metformin in combination with PI3K inhibitors resulted in a significant decrease in proliferation than either agent or metformin alone. While metformin exerts antineoplastic effects by activation of AMPK and decreased PI3K signaling, our data suggest that metformin may also disrupt localization of K-Ras and hence its signaling in an AMPK-independent manner. This has important implications in defining patients who may benefit from metformin in combination with other targeted agents, such as mTOR inhibitors.

Targeting AKT/mTOR Signaling Pathways During Murine Skin Tumor Promotion and the Impact of Dietary Energy Balance Manipulation

The prevalence of obesity has continued to rise over the last several decades in the United States lending to overall increases in risk for chronic diseases including many types of cancer. In contrast, reduction in energy consumption via calorie restriction (CR) has been shown to be a potent inhibitor of carcinogenesis across a broad range of species and tumor types. Previous data has demonstrated differential signaling through Akt and mTOR via the IGF-1R and other growth factor receptors across the diet-induced obesity (DIO)/CR spectrum. Furthermore, mTORC1 is known to be regulated directly via nutrient availability, supporting its role in the link between epithelial carcinogenesis and diet-induced obesity. In an effort to better understand the importance of mTORC1 in the context of both positive and negative energy balance during epithelial carcinogenesis, we have employed the use of specific pharmacological inhibitors, rapamycin (mTORC1 inhibitor) and metformin (AMPK activator) to target mTORC1 or various components of this pathway during skin tumor promotion. Two-stage skin carcinogenesis studies demonstrated that mTORC1 inhibition via rapamycin, metformin or combination treatments greatly inhibited skin tumor development in normal, overweight and obese mice. Furthermore, mechanisms by which these chemopreventive agents may be exerting their anti-tumor effects were explored. In addition, the effect of these compounds on the epidermal proliferative response was analyzed and drastic decreases in epidermal hyperproliferation and hyperplasia were found. Rapamycin also inhibited dermal inflammatory cell infiltration in a dose-dependent manner. Both compounds also blocked or attenuated TPA-induced signaling through epidermal mTORC1 as well as several downstream targets. In addition, inhibition of this pathway by metformin appeared to be, at least in part, dependent on AMPK activation in the skin. Overall, the data indicate that pharmacological strategies targeting this pathway offset the tumor-enhancing effects of DIO and may serve as possible CR mimetics. They suggest that mTORC1 contributes significantly to the process of skin tumor promotion, specifically during dietary energy balance effects. Exploiting the mechanistic information underlying dietary energy balance responsive pathways will help translate decades of research into effective strategies for prevention of epithelial carcinogenesis.

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

The hypotensive effect of acute and chronic AMP-activated protein kinase activation in normal and hyperlipidemic mice

AMP-activated protein kinase (AMPK) is present in the arterial wall and is activated in response to cellular stressors that raise AMP relative to ADP/ATP. Activation of AMPK in vivo lowers blood pressure but the influence of hyperlipidemia on this response has not been studied. ApoE-/- mice on high fat diet for 6 weeks and age-matched controls were treated with the AMPK activator, AICAR daily for two weeks. Under anesthesia, the carotid artery was cannulated for blood pressure measurements. Aortic tissue was removed for in vitro functional experiments and AMPK activity was measured in artery homogenates by Western blotting. ApoE-/- mice had significantly raised mean arterial pressure; chronic AICAR treatment normalized this but had no effect in normolipidemic mice, whereas acute administration of AICAR lowered mean arterial pressure in both groups. Chronic AICAR treatment increased phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase in normolipidemic but not ApoE-/- mice. In aortic rings, AMPK activation induced vasodilation and an anticontractile effect, which was attenuated in ApoE-/- mice. This study demonstrates that hyperlipidemia dysregulates the AMPK pathway in the arterial wall but this effect can be reversed by AMPK activation, possibly through improving vessel compliance.

Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.