Central Regulator Of Cellular Metabolism Research Articles

Mitochondrial Mutations in Cardiovascular Diseases: Preliminary Findings.

Background/Objectives: Mitochondria are the main organelles for ATP synthesis able to produce energy for several different cellular activities. Cardiac cells require high amounts of energy and, thus, they contain a high number of mitochondria. Consequently, mitochondrial dysfunction in these cells is a crucial factor for the development of cardiovascular diseases. Mitochondria constitute central regulators of cellular metabolism and energy production, producing approximately 90% of the cells' energy needs in the form of ATP via oxidative phosphorylation. The mitochondria have their own circular, double-stranded DNA encoding 37 genes. Any mitochondrial DNA sequence anomaly may result in defective oxidative phosphorylation and lead to cardiac dysfunction. Methods: In this study, we investigated the potential association between mitochondrial DNA mutation and cardiovascular disease. Cardiac tissue and serum samples were collected from seven patients undergoing coronary artery bypass grafting. Total DNA was extracted from cardiac muscle tissue specimens and serum and each sample was subjected to polymerase chain reaction (PCR) to amplify the NADH dehydrogenase 1 (ND1) gene, which is part of the mitochondrial complex I enzyme complex and was screened for mutations. Results: We identified one patient with a homoplasmic A to G substitution mutation in cardiac tissue DNA and two patients with heteroplasmic A3397G mutation in serum DNA. Specifically, amplicon sequence analysis revealed a homoplasmic A3397G substitution in the ND1 gene in a tissue sample of the patient with ID number 1 and a heteroplasmic mutation in A3397G in serum samples of patients with ID numbers 3 and 6, respectively. The A to G substitution changes the amino acid from methionine (ATA) to valine (GTA) at position 31 of the ND1 gene. Conclusions: The detection of this novel mutation in patients with coronary artery disease may contribute to our understanding of the association between mitochondrial dysfunction and the disease, implying that mitochondria may be key players in the pathogenesis of cardiovascular diseases.

The molecular basis of nutrient sensing and signalling by mTORC1 in metabolism regulation and disease.

The Ser/Thr kinase mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolism. As part of mTOR complex 1 (mTORC1), mTOR integrates signals such as the levels of nutrients, growth factors, energy sources and oxygen, and triggers responses that either boost anabolism or suppress catabolism. mTORC1 signalling has wide-ranging consequences for the growth and homeostasis of key tissues and organs, and its dysregulated activity promotes cancer, type 2 diabetes, neurodegeneration and other age-related disorders. How mTORC1 integrates numerous upstream cues and translates them into specific downstream responses is an outstanding question with major implications for our understanding of physiology and disease mechanisms. In this Review, we discuss recent structuraland functional insights into the molecular architecture of mTORC1and its lysosomal partners, which have greatly increased our mechanisticunderstanding of nutrient-dependent mTORC1 regulation. We also discuss the emerging involvement of aberrant nutrient-mTORC1 signalling in multiple diseases.

MTORC1 Deficiency Prevents the Development of MC903-Induced Atopic Dermatitis through the Downregulation of Type 2 Inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczema and itching. Recently, mTORC, a central regulator of cellular metabolism, has been reported to play a critical role in immune responses, and manipulation of mTORC pathways has emerged as an effective immunomodulatory drug. In this study, we assessed whether mTORC signaling could contribute to the development of AD in mice. AD-like skin inflammation was induced by a 7-day treatment of MC903 (calcipotriol), and ribosomal protein S6 was highly phosphorylated in inflamed tissues. MC903-induced skin inflammation was ameliorated significantly in Raptor-deficient mice and exacerbated in Pten-deficient mice. Eosinophil recruitment and IL-4 production were also decreased in Raptor deficient mice. In contrast to the pro-inflammatory roles of mTORC1 in immune cells, we observed an anti-inflammatory effect on keratinocytes. TSLP was upregulated in Raptor deficient mice or by rapamycin treatment, which was mediated by hypoxia-inducible factor (HIF) signaling. Taken together, these results from our study indicate the dual roles of mTORC1 in the development of AD, and further studies on the role of HIF in AD are warranted.

Polystyrene nanoplastics induce profound metabolic shift in human cells as revealed by integrated proteomic and metabolomic analysis.

Nanoplastics (NPLs) are widespread in our environment. However, their impacts on human health and precise toxicity mechanisms remain poorly understood. Here we studied the internalization, release, and cytotoxicity of polystyrene nanoplastics (PSNPs) using the renal tubular epithelial cell line HKC and human derived liver cell line HL-7702. We also employed an integrated proteomic and metabolomic approach to investigate the potential biological effects of PSNPs on HKC cells. The abundances of 4770 proteins and 100 metabolites were quantified, with 785 proteins and 17 metabolites detected with altered levels in response to PSNPs. Most of the differential proteins and metabolites were enriched in a variety of metabolic pathways, for example, glycolysis, citrate cycle, oxidative phosphorylation, and amino acid metabolism, suggesting the potential effects of NPLs on global cellular metabolism shift in human cells. The altered energy metabolism induced by PSNPs was further confirmed by a Seahorse analysis. Moreover, lysosomal distribution study and western blotting showed that mTORC1 signaling, a central regulator of cellular metabolism, was inhibited upon nanoplastic exposure, likely serving as the link between lysosome dysfunction and metabolic defects. Taken together, our findings systematically mapped the key molecular changes induced by PSNPs in human cells and provide comprehensive biological insights for the risk estimation of NPLs contamination.

The Clinical Effects of Nicotinamide Riboside on Inflammatory Parameters

ObjectivesNicotinamide adenine dinucleotide (NAD+) coenzymes are the central regulators of cellular metabolism. While the NAD + system is dysregulated by metabolic stress and age-related conditions, nicotinamide riboside (NR) augments NAD + levels and may contribute to the reduction of inflammation. As advancing age is often accompanied by chronically elevated inflammation (i.e., ‘inflammaging’), targeting chronic inflammation may be a viable strategy to preserve aspects of health and function. The objective of this study was to investigate the clinical evidence of NR's mitigation of inflammation in humans.MethodsWe systematically evaluated peer-reviewed, published clinical trials of oral NR supplementation that included analysis of inflammatory markers. There were no restrictions for age, sex, health status, or use of other bioactives. Key study details were assessed to compare experimental designs and results.ResultsWe identified 5 clinical trials of oral NR supplementation that measured inflammation. There was substantial heterogeneity in inflammatory endpoints evaluated. NR dose and treatment duration ranged from 1–2 g/day and 5-d– 6-wks, respectively. Participants varied in age (18–78 years) and health status. In one study, NR was given as part of a nutritional cocktail, while NR was the sole intervention in the remaining studies. Of the 3 studies assessing circulating inflammatory markers, two investigated IL-6 and reported significant reductions in its levels with NR treatment, while effects on MCP-1 and TNF-α were inconsistent. NR significantly lowered gene expression of IL-6 and IL-18, but not NLRP3 or IL-1B in peripheral blood mononuclear cells in one study. Another study documented significant reductions in the gene expression of IFNB and CXCL10, but no change to TNFA expression, in the monocytes of the NR-treated group. Moreover, NR treatment led to lower concentrations of IFNβ, but not TNF-α, in monocytes.\\ConclusionsPreliminary evidence suggests that NR may improve some inflammatory measures, with potential for more robust effects among populations with compromised NAD + and higher inflammation status, e.g., elderly or diseased. More work is needed to clarify which inflammatory markers are targeted by NR, and the extent to which they are further modified by dose and participant demographics.Funding SourcesN/A.

Complete Genome Sequence of Lactobacillus hilgardii LMG 7934, Carrying the Gene Encoding for the Novel PII-Like Protein PotN.

Lactic acid bacteria are widespread in various ecological niches with the excess of nutrients and have reduced capabilities to adapt to starvation. Among more than 280 Lactobacillus species known to the date, only five, including Lactobacillus hilgardii, carry in their genome the gene encoding for PII-like protein, one of the central regulators of cellular metabolism generally responding to energy- and carbon-nitrogen status in many free-living Bacteria, Archaea and in plant chloroplasts. In contrast to the classical PII encoding genes, in L.hilgardii genome the gene for PII homologue is located within the potABCD operon, encoding the ABC transporter for polyamines. Based on the unique genetic context and low sequence identity with genes of any other so-far characterized PII subfamilies, we termed this gene potN (Pot-protein, Nucleotide-binding). The second specific feature of L.hilgardii genome is that many genes encoding the proteins with similar function are present in two copies, while with low mutual identity. Thus, L.hilgardii LMG 7934 genome carries two genes of glutamine synthetase with 55% identity. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. Together with the relative large genome of L.hilgardii as compared to other Lactobacilli (2.771.862bp vs ~ 2.2 Mbp in median), these data suggest significant re-arrangements of the genome and a wider range of adaptive capabilities of L.hilgardii in comparison to other bacteria of the genus Lactobacillus.

Identify AMPK-Mediated Autophagy and Oxphos As a Novel Metabolic Vulnerability for Targeted Therapy in Mantle Cell Lymphoma

Introduction: Metabolic reprogramming promotes cancer cell growth, metastasis, and therapeutic resistance in a multitude of cancers. Mantle cell lymphoma (MCL) is a rare and aggressive hematopoietic malignancy that exhibits dramatic alterations in the cellular metabolism, especially in advanced stages and at the time of relapse. Unchecked pro-tumor growth signaling and multidrug therapy induce genomic instability and metabolic stress. To sustain tumor survival, MCL cancer cells upregulate multiple essential metabolic pathways that include mitochondria biogenesis, OXPHOS, glutaminolysis, cytoprotective autophagy, and fatty acid β-oxidation. While we recently have demonstrated that OXPHOS is upregulated and mediates resistance to ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, in MCL, the underlying mechanism remains to be elucidated. Cytoprotective autophagy is known to be induced in chronic hypoxic microenvironments such as bone marrow and secondary lymphoid organs, and it in turn contributes to drug resistance and promotes MCL tumor survival. AMPK, a central regulator of cellular metabolism, is activated when MCL cells are challenged with metabolic stress. Consistently, AMPKα1 is highly activated in a subset of MCL at the leukemic phase. AMPK dependence and metabolic reprogramming represent vulnerabilities that could be potentially targeted for MCL treatment. Methods: Primary MCL biopsies, apheresis, or blood specimens, as well as MCL cell lines, were used for metabolic and functional analyses. Metabolomics profiling was carried out using Liquid Chromatography Mass Spectrometry (LC-MS). Mitochondrial membrane potential was determined by staining MCL cells with MitoStatus or TMRE and flow cytometry analysis. OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were determined by Seahorse metabolic flux analysis. Autophagy flux was monitored with tandem mRFP-EGFP-LC3 fluorescence reporter analysis. Other analyses included western blotting, real-time qPCR, and cell viability assay (Cell Titer-Glo). Pharmacological inhibitors were used for the down-modulation of ULK1, AMPK, autophagy, and OXPHOS. Results: Metabolomics profiling of steady-state levels of TCA metabolites showed significant increases in levels of α-KG, glutamate, malate, and fumarate, indicating upregulated glutaminolysis in subsets of MCL that are recurrent or refractory to targeted therapeutics including ibrutinib. Functional analysis illustrates that glutamine deprivation leads to reduced ATP and cell viability in a subset of MCL, while pharmacological inhibition of glutamine metabolism leads to reduced mitochondria membrane potential and increased cellular apoptosis. Seahorse metabolic flux analysis revealed enhanced OXPHOS activity in ibrutinib-resistant MCL cells as indicated by increased OCR. Inhibition of OXPHOS or glutamine metabolism impairs OCR and subsequently induces dose-dependent cellular apoptosis. Further, autophagy flux is increased in the same MCL cells with mRFP-EGFP-LC3 as a reporter. IHC and western blot analysis demonstrated that AMPKα1 is highly activated in a subset of MCL at the leukemic phase, concomitant with an increased NADP:NADPH and AMP:ATP ratio, indicating energy stress. AMPK activation promotes cytoprotective autophagy, and AMPK blockade suppresses autophagy, mitochondria biogenesis, and activity (OCR and ATP production), ultimately inhibiting MCL proliferation under metabolic stress. Conclusions: Our results demonstrate that AMPK activation promotes cytoprotective autophagy, glutaminolysis, and OXPHOS in established MCL, thus representing unique metabolic vulnerabilities that could be potentially targeted for MCL treatment. As AMPKα1 inhibitors are underexplored, it is critical to identify and develop more potent and specific inhibitors of the kinase and define the in vivo functions of its activation in certain advanced cancers. Similarly, pharmacological blockade of the dysregulated autophagy, glutaminolysis, and OXPHOS can be extensively exploited for treatment of MCL patients, especially those at advanced stages of the disease. Disclosures Wang: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; MoreHealth: Consultancy, Equity Ownership; Acerta Pharma: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Guidepoint Global: Consultancy; BioInvent: Consultancy, Research Funding; VelosBio: Research Funding; Loxo Oncology: Research Funding; Celgene: Honoraria, Research Funding; Juno Therapeutics: Research Funding; Aviara: Research Funding; Dava Oncology: Honoraria.

AMP-Activated Protein Kinase Regulates Circadian Rhythm by Affecting CLOCK in Drosophila.

The circadian clock organizes the physiology and behavior of organisms to their daily environmental rhythms. The central circadian timekeeping mechanism in eukaryotic cells is the transcriptional-translational feedback loop (TTFL). In the Drosophila TTFL, the transcription factors CLOCK (CLK) and CYCLE (CYC) play crucial roles in activating expression of core clock genes and clock-controlled genes. Many signaling pathways converge on the CLK/CYC complex and regulate its activity to fine-tune the cellular oscillator to environmental time cues. We aimed to identify factors that regulate CLK by performing tandem affinity purification combined with mass spectrometry using Drosophila S2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. We identified SNF4Aγ, a homolog of mammalian AMP-activated protein kinase γ (AMPKγ), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPKα and two regulatory subunits, AMPKβ and AMPKγ, directly phosphorylated purified CLK in vitro Locomotor behavior analysis in Drosophila revealed that knockdown of each AMPK subunit in pacemaker neurons induced arrhythmicity and long periods. Knockdown of AMPKβ reduced CLK levels in pacemaker neurons, and thereby reduced pre-mRNA and protein levels of CLK downstream core clock genes, such as period and vrille Finally, overexpression of CLK reversed the long-period phenotype that resulted from AMPKβ knockdown. Thus, we conclude that AMPK, a central regulator of cellular energy metabolism, regulates the Drosophila circadian clock by stabilizing CLK and activating CLK/CYC-dependent transcription.SIGNIFICANCE STATEMENT Regulation of the circadian transcription factors CLK and CYC is fundamental to synchronize the core clock with environmental changes. Here, we show that the AMPKγ subunit of AMPK, a central regulator of cellular metabolism, copurifies with the CLK/CYC complex in Drosophila S2 cells. Furthermore, the AMPK holoenzyme directly phosphorylates CLK in vitro This study demonstrates that AMPK activity regulates the core clock in Drosophila by activating CLK, which enhances circadian transcription. In mammals, AMPK affects the core clock by downregulating circadian repressor proteins. It is intriguing to note that AMPK activity is required for core clock regulation through circadian transcription enhancement, whereas the target of AMPK action is different in Drosophila and mammals (positive vs negative element, respectively).

Hyperactivation of mTORC1 disrupts cellular homeostasis in cerebellar Purkinje cells

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.

Novel roles of mechanistic target of rapamycin signaling in regulating fetal growth†.

Mechanistic target of rapamycin (mTOR) signaling functions as a central regulator of cellular metabolism, growth, and survival in response to hormones, growth factors, nutrients, energy, and stress signals. Mechanistic TOR is therefore critical for the growth of most fetal organs, and global mTOR deletion is embryonic lethal. This review discusses emerging evidence suggesting that mTOR signaling also has a role as a critical hub in the overall homeostatic control of fetal growth, adjusting the fetal growth trajectory according to the ability of the maternal supply line to support fetal growth. In the fetus, liver mTOR governs the secretion and phosphorylation of insulin-like growth factor binding protein 1 (IGFBP-1) thereby controlling the bioavailability of insulin-like growth factors (IGF-I and IGF-II), which function as important growth hormones during fetal life. In the placenta, mTOR responds to a large number of growth-related signals, including amino acids, glucose, oxygen, folate, and growth factors, to regulate trophoblast mitochondrial respiration, nutrient transport, and protein synthesis, thereby influencing fetal growth. In the maternal compartment, mTOR is an integral part of a decidual nutrient sensor which links oxygen and nutrient availability to the phosphorylation of IGFBP-1 with preferential effects on the bioavailability of IGF-I in the maternal-fetal interface and in the maternal circulation. These new roles of mTOR signaling in the regulation fetal growth will help us better understand the molecular underpinnings of abnormal fetal growth, such as intrauterine growth restriction and fetal overgrowth, and may represent novel avenues for diagnostics and intervention in important pregnancy complications.

Lysosomes Mediate Benefits of Intermittent Fasting in Cardiometabolic Disease: The Janitor Is the Undercover Boss.

Adaptive responses that counter starvation have evolved over millennia to permit organismal survival, including changes at the level of individual organelles, cells, tissues, and organ systems. In the past century, a shift has occurred away from disease caused by insufficient nutrient supply toward overnutrition, leading to obesity and diabetes, atherosclerosis, and cardiometabolic disease. The burden of these diseases has spurred interest in fasting strategies that harness physiological responses to starvation, thus limiting tissue injury during metabolic stress. Insights gained from animal and human studies suggest that intermittent fasting and chronic caloric restriction extend lifespan, decrease risk factors for cardiometabolic and inflammatory disease, limit tissue injury during myocardial stress, and activate a cardioprotective metabolic program. Acute fasting activates autophagy, an intricately orchestrated lysosomal degradative process that sequesters cellular constituents for degradation, and is critical for cardiac homeostasis during fasting. Lysosomes are dynamic cellular organelles that function as incinerators to permit autophagy, as well as degradation of extracellular material internalized by endocytosis, macropinocytosis, and phagocytosis. The last decade has witnessed an explosion of knowledge that has shaped our understanding of lysosomes as central regulators of cellular metabolism and the fasting response. Intriguingly, lysosomes also store nutrients for release during starvation; and function as a nutrient sensing organelle to couple activation of mammalian target of rapamycin to nutrient availability. This article reviews the evidence for how the lysosome, in the guise of a janitor, may be the "undercover boss" directing cellular processes for beneficial effects of intermittent fasting and restoring homeostasis during feast and famine. © 2018 American Physiological Society. Compr Physiol 8:1639-1667, 2018.

5'-Adenosine monophosphate-activated protein kinase: A potential target for disease prevention by curcumin.

Curcumin (diferuloylmethane), a yellowish agent extracted from turmeric, is a bioactive compound known for its anti-inflammatory, antiproliferative, antidiabetic, and anticancer activities. Multiple lines of evidence have indicated that curcumin regulates several regulatory proteins in the cellular signal transduction pathway. AMP-activated protein kinase (AMPK) is one of the central regulators of cellular metabolism and energy homeostasis, which is activated in response to increasing cellular adenosine monophosphate/adenosine triphosphate ratio. AMPK plays a critical role in regulating growth and reprogramming metabolism and is linked to several cellular processes including apoptosis and inflammation. Recently, it has been demonstrated that AMPK is a new molecular target affected by curcumin and its derivatives. In this review, we discuss recent findings on the targeting of AMPK signaling by curcumin and the resulting impact on the pathogenesis of proinflammatory diseases. We also highlight the therapeutic value of targeting AMPK by curcumin in the prevention and treatment of proinflammatory diseases, including cancers, atherosclerosis, and diabetes.

MTORc1 activity is necessary and sufficient for phosphorylation of eNOSS1177.

Nitric oxide, produced by eNOS, plays critical roles in the regulation of vascular function and maintenance. Chronic PI3K signaling has recently been associated with vascular malformations. A well described substrate downstream of PI3K signaling is eNOS. Another critical downstream target of PI3K is the metabolic regulator, mTORc1. The relationship between mTORc1 and eNOS regulation, has not been determined. We generated cells with manipulated PI3K signaling by expressing the activating mutation, PIK3CAH1047R, or knocking down PTEN expression. We investigated eNOSS1177 phosphorylation, a major activating regulatory site, following mTORC1 inhibition. We also tested the sufficiency of mTORc1 activation to stimulate eNOSS1177 phosphorylation. Our data indicate mTORc1 activity is required for the phosphorylation of eNOSS1177, even in the presence of robust AKT activation. Moreover, we found that expression of RHEB, which functions in the absence of AKT activation to activate mTORc1, is sufficient to phosphorylate this site. Our data indicate that mTORc1, rather than AKT, may be the critical determinant of eNOSS1177 phosphorylation. As mTORc1 is a central regulator of cellular metabolism, the finding that this regulatory complex can directly participate in the regulation of eNOS provides new insights into metabolic uncoupling and vascular disease that often accompanies diabetes, high fat diets, and aging.

Regulation of mTOR, Metabolic Fitness, and Effector Functions by Cytokines in Natural Killer Cells.

The control of cellular metabolism is now recognized as key to regulate functional properties of immune effectors such as T or Natural Killer (NK) cells. During persistent infections or in the tumor microenvironment, multiple metabolic changes have been highlighted in T cells that contribute to their dysfunctional state or exhaustion. NK cells may also undergo major phenotypic and functional modifications when infiltrating tumors that could be linked to metabolic alterations. The mammalian target of rapamycin (mTOR) kinase is a central regulator of cellular metabolism. mTOR integrates various extrinsic growth or immune signals and modulates metabolic pathways to fulfill cellular bioenergetics needs. mTOR also regulates transcription and translation thereby adapting cellular pathways to the growth or activation signals that are received. Here, we review the role and regulation of mTOR in NK cells, with a special focus on cytokines that target mTOR such as IL-15 and TGF-β. We also discuss how NK cell metabolic activity could be enhanced or modulated to improve their effector anti-tumor functions in clinical settings.

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

BackgroundGlycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.MethodsThe expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.ResultsExpression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.ConclusionNSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.

The AMP-activated protein kinase (AMPK) and cancer: Many faces of a metabolic regulator

The AMP-activated protein kinase (AMPK) is a central regulator of cellular metabolism and energy homeostasis in mammalian tissues. Pertinent to cancer biology is the fact that AMPK is situated in the center of a signaling network involving established tumor suppressors including LKB1, TSC2 and p53. However, recent research suggests that AMPK can exert pro- or anti-tumorigenic roles in cancer depending on context. Loss of AMPK activity has been observed in several tumor types, and can cooperate with oncogenic drivers to reprogram tumor cell metabolism and enhance cell growth and proliferation. However, AMPK activation can also provide a growth advantage to tumor cells by regulating cellular metabolic plasticity, thus providing tumor cells the flexibility to adapt to metabolic stress. Here we discuss the contextual nature of the “two faces” of AMPK in cancer, and discuss the rationale and context for employing AMPK activators versus inhibitors for cancer therapy.

Mitofusins: Mighty Regulators of Metabolism

Mitochondria are central regulators of cellular metabolism but how their function in a subset of cells affects whole-body energy balance is less understood. Two studies in this issue of Cell identify how diet-dependent modulation of mitochondrial fusion in specific neuronal circuits impact the metabolic status of an animal.

Regulation of Cytokine Signal Transduction in Hematopoietic Stem Cells by Mammalian Target of Rapamycin

Abstract 1239 Background:Hematopoiesis is a coordinated process in which hematopoietic stem cells (HSCs) undergo self-renewal and differentiation to produce multilineage blood cells. Maintenance of the HSC compartment has been shown to require regulation by a diverse profile of cytokines including members of the receptor tyrosine kinase (RTK) superfamily, with thrombopoietin (TPO) identified as a central regulator of HSC fate. HSC regulation is also mediated by serine-threonine kinases, such as transforming growth factor-beta (TGF-beta). Improved understanding of the mechanisms regulating HSC proliferation, self-renewal, and quiescence is essential for improving transplantation, ex vivo expansion, and other therapeutic applications of these cells.Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism, nutrient sensing and autophagy. mTOR has also emerged as a central regulator of HSC proliferation and self-renewal. Mouse models of mTOR hyperactivation demonstrate an imbalance of HSC proliferation and self-renewal leading to exhaustion of repopulating cells in a rapamycin-dependent manner, and pretreatment of human cord blood CD34+ cells with rapamycin has been shown to increase engraftment in immunodeficient mice. However, the mechanisms responsible for mTOR regulation of HSCs remain poorly understood.Here we show that inhibition of mTOR by rapamycin significantly enhances signal transduction through both the TPO and TGF-beta pathways by inhibiting receptor endocytosis, resulting in increased cell surface levels of growth factor receptors and enhanced engraftment in a mouse model of prenatal HSC transplantation. Methods:We employed primary and immortalized HSCs, as well as 293T cells stably overexpressing the thrombopoietin receptor, to study the effects of mTOR inhibition. Cells were treated with rapamycin for 72 hours prior to growth factor stimulation, and activation of signaling proteins were measured by immunoblotting and immunofluorescence studies. Surface receptor levels were determined by biotinylation and streptavidin immunoprecipitation. Clathrin- and caveolin-dependent endocytosis was inhibited by potassium depletion and nystatin treatment, respectively. Bone marrow transplantation studies were preformed in a well-characterized mouse model of in utero hematopoietic cell transplantation at 14 days gestational age, with engraftment quantified at 96 hours and 2 weeks postnatally. Results:Rapamycin treatment resulted in enhanced signal transduction through both the TPO and TGF-beta pathways, with significantly increased levels of phosphorylation and nuclear translocation of MAPK and Smad proteins respectively (Figure 1A). Biotinylation studies revealed increased levels of cell surface receptors following mTOR inhibition. Pharmacologic disruption of endocytosis was found to ablate the effects of rapamycin treatment, with equivalent and robust activation of MAPK and Smad proteins in rapamycin-treated and untreated cells (Figure 1B). To test the hypothesis that enhanced cytokine signaling would result in an optimal profile of HSC function, in utero transplantation of rapamycin-treated primary bone marrow cells was performed. Rapamycin treatment resulted in enhanced HSC engraftment levels at 96 hours post-transplantation (4.2 +/− 0.6% vs 23.8 +/− 2.2%, p<0.005), and postnatal chimerism levels assessed at 2 weeks of age were similarly found to be significantly higher in the rapamycin-treatment group compared to controls (2.2 +/− 0.6% vs 29.8 +/− 10.7%, p<0.005). Conclusions:These results support a role for mTOR in modulating HSC signal transduction by regulation of cytokine receptor endocytosis, resulting in enhanced levels of engraftment following transplantation. The observed effects of mTOR inhibition on multiple diverse cytokine profiles suggests a central mechanism of regulation of intracellular trafficking, which may be mediated at the level of formation of early endosomal and autophagosome complexes. Further studies will delineate receptor fate following internalization by these pathways. These studies will ultimately improve understanding of how the manipulation of multiple signaling circuits may be optimized to control self-renewal and quiescence compatible for in vitro expansion and transplantation of HSCs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HIF1α–dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells

Upon antigen stimulation, the bioenergetic demands of T cells increase dramatically over the resting state. Although a role for the metabolic switch to glycolysis has been suggested to support increased anabolic activities and facilitate T cell growth and proliferation, whether cellular metabolism controls T cell lineage choices remains poorly understood. We report that the glycolytic pathway is actively regulated during the differentiation of inflammatory T(H)17 and Foxp3-expressing regulatory T cells (T(reg) cells) and controls cell fate determination. T(H)17 but not T(reg) cell-inducing conditions resulted in strong up-regulation of the glycolytic activity and induction of glycolytic enzymes. Blocking glycolysis inhibited T(H)17 development while promoting T(reg) cell generation. Moreover, the transcription factor hypoxia-inducible factor 1α (HIF1α) was selectively expressed in T(H)17 cells and its induction required signaling through mTOR, a central regulator of cellular metabolism. HIF1α-dependent transcriptional program was important for mediating glycolytic activity, thereby contributing to the lineage choices between T(H)17 and T(reg) cells. Lack of HIF1α resulted in diminished T(H)17 development but enhanced T(reg) cell differentiation and protected mice from autoimmune neuroinflammation. Our studies demonstrate that HIF1α-dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of T(H)17 and T(reg) cells.

Akt enzyme: new therapeutic target in cancer and diabetes?

Alteration of apoptotic processes plays a central role in the development and progression of several chronic disorders. Proteins responsible for the regulation of apoptosis are therapeutic targets; these include the Akt enzyme. Akt enzyme is expressed in most cell types. Akt activation is regulated by growth factors, insulin, and also environmental factors as altered oxygen tension and high temperature. Akt is a central regulator of cellular metabolism and survival. Akt function is reportedly altered in some disorders. An increased activity of Akt has been described in prostate, breast, colon, and pancreatic cancer, as well as in hematological malignancies. Akt is also a factor in the pathomechanism of diabetes as it determines beta-cell apoptosis of Langerhans islets and insulin sensitivity of the cells. Several studies revealed that some of the marketed drugs including statins, thiazolidinediones and ACE inhibitors modulate Akt activity. There are efforts to develop specific Akt inhibitors that may improve the efficacy of chemotherapy. Triciribine and perifosine are two Akt inhibitors in developmental phase 1 and 2 that may improve survival in breast cancer, pancreas cancer, gastrointestinal stroma tumor, sarcoma and melanoma, and in hematological malignancy.