Browsing by Subject "Metabolism"
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Item Open Access A tale of two metallophosphatases: biochemical and functional characterization of novel substrates of PP1 and MESH1(2017) Rose, Joshua StevenAddition and removal of phosphate is an important post-translational modification involved in cellular signaling. The enzymes responsible for removing this phosphorylation mark, called phosphatases, play a vital role in the cellular decision making processes. In this work we discuss two discoveries, a novel enzyme for a known signaling function involving control of transcription and a novel target for an important cellular stress response enzyme.
In the first project we sought to determine a novel enzyme responsible for dephosphorylating the C-terminal domain of RNA polymerase II. This domain serves as a vital signaling platform for transcription of mammalian genes, with the ability to recruit cofactors that bind to specific patterns of phosphorylation throughout its repeating amino acid sequence. Using a functional assay for phosphatase activity at the Thr4 position we biochemically isolated the unknown enzyme and identified it as PP1 and validated its function in vitro and in vivo.
The second phosphatase studied in this dissertation is MESH1—a mammalian ortholog of the bacterial stringent response protein SpoT that dephosphorylates ppGpp. Because ppGpp is absent in mammalian cells MESH1 lacks a viable target. We established NADPH as a substrate of MESH1 biochemically and corroborated these results by determining the substrate bound structure. Our results reveal a novel regulatory role of MESH1 in a pathway that resembles the bacterial stringent response.
Item Open Access Aerobic Training-Induced Host Changes Alter Breast Cancer Cell Phenotypes and Tumor Progression(2015) Glass, OliverA growing number of studies have investigated the role of exercise both during and after a breast cancer diagnosis. Observational data suggests that regular endurance exercise is associated with a 20-50% reduction in cancer-specific mortality in women diagnosed with early stage breast cancer, compared to inactive women; however it is unclear whether there is a differential association across breast cancer subtypes. As a pre-requisite to guide future large phase II/III clinical trials, there is a critical need to confirm the biological plausibility of the exercise association in breast cancer patients as well as elucidate the underlying mechanisms of action via utilization of preclinical models.
In the present study we investigated the systemic effects of prescribed aerobic training in cancer patients and the direct impact on breast cancer cell subtype phenotypes. In order to test the in vivo significance, we interrogated aerobic training effects on breast cancer progression and tumor biology using syngeneic breast cancer mouse models.
Our results suggest that aerobic training may alter the host availability of pro-inflammatory and growth factor cytokines in patients with solid tumors. Modulation of systemic effectors in breast cancer patients compared to controls causes a differential phenotypic response on breast cancer cell subtypes. In vivo, aerobic training has a differential response on breast tumor progression compared to controls that is mediated by Hif1-α and metabolic reprogramming of breast cancer cells.
Item Open Access Autophagy in Metabolism, Cell Death, and Leukemogenesis(2011) Altman, Brian JamesTissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Cancer cells, however, can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells are deprived sufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and cells activate the self-digestive process of autophagy, which clears damaged organelles and provides degradation products as an alternate fuel to support mitochondrial metabolism. Importantly, loss of growth signals can also lead to apoptosis mediated through Bcl-2 family proteins, and Bcl-2 has been reported to interfere with autophagy, potentially disrupting a key nutrient source just as glucose uptake becomes limiting. Since autophagy may support survival or lead to death depending on context, the role of this pathway in apoptosis-competent growth factor deprived cells remains unclear.
In this thesis, I examine the interactions of autophagy with Bcl-2 family proteins and apoptosis upon inhibition of growth signals in hematopoietic cells. In contrast to other studies, I found autophagy was rapidly induced in growth factor deprived cells regardless of Bcl-2 or Bcl-xL expression, and this led to increased production of fatty acids and amino acids for metabolism. While these data suggested autophagy may play a key role to support metabolism of growth factor deprived cells, provision of exogenous pyruvate or lipids as alternate fuel had little affect on cell survival. Instead, I found that autophagy modulated cell stress pathways and Bcl-2 family protein expression in a context specific fashion to impact cell fate.
My results show that autophagy's effect on cell survival is dependent on its level of induction within a cell. I observed that partial suppression of autophagy protects cells from stress and induction of pro-apoptotic Bcl-2 family expression, while complete inhibition of autophagy enhances stress and is pro-apoptotic. In experiments using shRNAi to partially suppress autophagy, I found increased survival upon growth factor deprivation in several different types of cells expressing anti-apoptotic Bcl-2 or Bcl-xL, indicating that autophagy promoted cell death in these instances. Cell death was not autophagic, but apoptotic, and relied on direct Chop-dependent transcriptional induction of the pro-apoptotic Bcl-2 family protein Bim. In contrast, complete acute disruption of autophagy through conditional Cre-mediated excision of the autophagy-essential gene Atg3 led to p53 phosphorylation, upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma, and rapid cell death of cells the presence or absence of growth factor. Importantly, transformed BCR-Abl-expressing cells had low basal levels of autophagy but were highly dependent on this process. Deletion of Atg3 or treatment with chemical autophagy inhibitors led to rapid apoptosis, and BCR-Abl expressing cells were unable to form leukemia in mice in without autophagy. Together, my data demonstrate a dual role for autophagy in cell survival or cell death and suggest that the level of autophagy in a cell is critical in determining its role in apoptosis and cell fate. Ultimately, these results may help to determine future approaches to modulate autophagy in cancer therapy.
Item Open Access Coupling of the Yeast Metabolic Cycle and the Cell Division Cycle in Populations and Single Cells(2017) Burnetti, Anthony JBiological oscillators are ubiquitous in living systems. They allow cellular processes to anticipate and act in synchrony with regular events in the outside world (such as the day/night cycle), or they ensure that processes occur in a particular order. Living things typically contain multiple oscillators, which can often couple to each other and influence each other's timing and function. The purpose of this thesis has been to investigate the relationship between two coupled oscillators in \textit{Saccharomyces cerevisiae}: the yeast metabolic cycle and the cell division cycle. I have focused on two key questions: what is the biological significance of their coupling, and is one oscillator dominant in its interaction with the other?
First, I investigated the temporal relationship between the cell division cycle and metabolic shifts that occur during the metabolic cycle across diverse yeast strains. I showed that a particular cell cycle event (DNA replication) was consistently delayed relative to a metabolic event (entry into the high oxygen consumption phase). This suggested that an earlier cell cycle event (Start and commitment to the cell cycle) was tied to the onset of high oxygen consumption. Second, I used fluorescent probes to examine the relationship between the metabolic cycle and the commitment to cell cycle progression at single-cell resolution. This revealed that cells enter high oxygen consumption phase of the metabolic cycle before passing Start, supporting a model of metabolic cycle/cell division cycle coupling in which the shorter metabolic cycle controls cell cycle commitment, likely via modulation of cell size thresholds.
Item Embargo Defining MAP4K3-mediated Signaling Pathways That Regulate mTORC1 Activation and Beyond(2023) Branch, Mary RoseGerminal center kinases (GCKs) belong to the mammalian Ste20-like family of serine/threonine kinases and participate in various signaling pathways needed to regulate a wide range of cellular activities. GCK-like kinase (GLK), also known as MAP4K3, belongs to the MAP kinase kinase kinase kinase (MAP4K) family of proteins and has recently been established as a key node in the amino acid response pathway and putative nutrient sensing regulator in cells, as it is required for the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1)—a central regulator of cell growth and metabolism. The precise mechanism(s) by which MAP4K3 activates mTORC1 under conditions of amino acid satiety, however, are undefined. Recent studies in the La Spada lab suggest MAP4K3 activates mTORC1 by phosphorylating the NAD-dependent deacetylase sirtuin 1 (Sirt1) and subsequently, inhibiting the LKB1-AMPK pathway—a pathway that suppresses mTORC1 activation during starvation. MAP4K3 has additionally been linked to the regulation of cellular stress responses, autophagy, growth, survival, and organismal lifespan through largely unknown pathways. My working hypothesis is that MAP4K3 serves as an amino acid sensor and activates mTORC1 through phosphorylation of Sirt1 and subsequent inhibition of the mTORC1-suppressing Sirt1-LKB1-AMPK pathway under conditions of amino acid satiety and engages different biological pathways by virtue of its protein interacting partners to control critical cellular processes involved in cell growth, survival, and lifespan. In study 1, I used amino acid depletion/restimulation experiments and phospho mass spectrometry to establish a direct link between MAP4K3 and the Sirt1-LKB1-AMPK pathway and determines that Sirt1 is phosphorylated at Threonine 344 (T344) in a MAP4K3- and amino acid-dependent manner. Furthermore, I showed that phosphorylation of T344 inhibits Sirt1 and is sufficient to restore amino acid-dependent mTORC1 activation in cells lacking MAP4K3. To elucidate additional pathways regulated by MAP4K3, in study 2, I sought to discover novel MAP4K3 interacting partners by integrating proteomics interactome data and phosphoproteomics data followed by validation studies in cells. Experiments from these studies indicate a novel role for MAP4K3 in regulating DNA double-strand break (DSB) sensing and repair in the nucleus, mTOR localization to the lysosome through the GATOR2 complex, and endocytosis. Recent discoveries regarding the important role for MAP4K3 in nutrient sensing through mTORC1 activation and other cellular activities, including cell growth, autophagy, and survival are significant because deregulation of these cellular processes has been implicated in aging, as well as a wide array of human diseases including cancer, immunological disorders, and neurodegeneration. This dissertation, thus, sheds light on the molecular mechanisms by which MAP4K3 regulates these processes and provides significant insight into the modulation of these pathways in health and disease states.
Item Open Access Digital Environmental Metabolisms: An Ecocritical Project of the Digital Environmental Humanities(2017) Gould, Amanda StarlingBy combining literary, ecocritical, and media techniques with a mindfulness of the environment, “Digital Environmental Metabolisms: An Ecocritical Project of the Digital Environmental Humanities” contributes to the urgent task of re-orienting media theory toward environmental concerns. It is informed by the premise that, in our present Anthropocenic age defined by humans acting as a geophysical force, human bodies, cultural technologies, and the earth are intersecting material practices. I argue this intersectionality is neither cyborgian nor posthuman, as some media scholars insist, but is something far more natural: it is a metabolic relationship wherein each system is inherently implicated in the perpetuation of the others. Through a series of chapters that dispense with standard maps of cyberspace and the social network replacing them with a digital geography of wires, workers, warehouses, and waste, this project shifts the media theoretical focus from one grounded in computation to one fully rooted in the earth. Unlike others, like those mentioned here within, who are contributing to what may be called an emerging environmental media studies, I offer several practical and theoretical interventions, including Permaculture and Ecocritical Digital Humanities, that are capable of moving us toward more sustainable digital practice and a more robust Anthropocene Humanities.
Item Open Access Dynamic Regulation of Metabolism in Archaea(2015) Todor, HoriaThe regulation of metabolism is one of the key challenges faced by organisms across all domains of life. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. Although much is known about the regulation of metabolism in the bacteria and the eukaryotes, relatively little is known about the regulation of metabolism in archaea. Previous work identified the winged helix-turn-helix transcription factor TrmB as a major regulator of metabolism in the model archaeon Halobacterium salinarum. TrmB was found to bind to the promoter of 113 genes in the absence of glucose. Many of these genes encode enzymes involved in metabolic processes, including central carbon metabolism, purine synthesis, and amino acid degradation. Although much is known about TrmB, it remains unclear how it dynamically regulates its ~100 metabolic enzyme-coding gene targets, what the effect of transcriptional regulation is on metabolite levels, and why TrmB regulates so many metabolic processes in response to glucose. Using dynamic gene expression and TrmB-DNA binding assays, we found that that TrmB functions alone to regulate central metabolic enzyme-coding genes, but cooperates with various regulators to control peripheral metabolic pathways. After determining the temporal pattern of gene expression changes and their dependence on TrmB, we used dynamic metabolite profiling to investigate the effects of transcriptional changes on metabolite levels and phenotypes. We found that TrmB-mediated transcriptional changes resulted in substantial changes in metabolite levels. Additionally, we showed that mis-regulation of genes encoding enzymes involved in gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low PRPP levels, which cause a metabolic block in de novo purine synthesis that is partially responsible for the growth defect of the ΔtrmB mutant strain. Finally, using a series of quantitative phenotyping experiments, we showed that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Because S-layer glycosylation is proportional to growth, we hypothesize that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Taken together, our results suggest that TrmB is a global regulator of archaeal metabolism that works in concert with other transcription factors to regulate diverse metabolic pathways in response to nutrients and growth rate.
Item Open Access Effects of Proximal Tubule Angiotensin II Signaling on Energy Metabolism in the Kidney(2017-12-12) Jimenez Contreras, FabianChronic kidney disease (CKD) affects over 26 million adults in the United States, thus it is imperative that we deduce more about the pathogenesis of the disease. CKD is generally multi-factorial, and loss of renal function can result from a number of diseases and pathologic processes. For example, propagation of kidney injury and renal fibrosis can result from abnormal regulation of energy metabolism in kidney cells. In renal proximal tubule epithelial cells, a key segment of the nephron, fatty acids are a major fuel source. As the proximal tubule is responsible for the bulk of sodium reabsorption by the kidney, maintaining adequate energy balance is crucial to this function; therefore, alterations in fatty acid oxidation in the renal proximal tubule may lead to renal dysfunction. Our hypothesis is that angiotensin II (Ang II) signaling, a major effector of the powerful renin-angiotensin system (RAS), alters fatty acid oxidation and this becomes exaggerated in states of renal injury such as hypertension and diabetes where the RAS can be dysregulated. Therefore, we sought to explore the metabolic changes linked to Ang II signaling in the renal proximal tubule. Increased levels of Ang II have previously been shown to induce renal fibrosis and hypertension. For our studies, we used a novel mouse line, one lacking AT1a receptors in renal proximal tubule cells (PTKO mice) and expected that the lack of AT1a receptors helps to maintain normal fatty acid oxidation in disease states. To model pathology which might stress the renal proximal tubule cells, we induced two diseases: hypertension, by infusing Ang II via osmotic mini pumps and diabetes, by employing a genetic model of type 1 diabetes, the Akita model. Our major outcome was the assessment of gene expression of several key metabolic pathways, using a quantitative PCR analysis of samples from mouse renal cortex, which is rich in proximal tubules. We aimed to measure genetic biomarkers in the fatty acid oxidation pathway, glucose oxidation pathway, markers of renal injury and fibrosis. These studies demonstrate how two clinically-relevant diseases influence metabolism in the kidney and how leveraging the RAS may lead to solutions against this disruption, and potentially alter CKD progression.Item Open Access Exploiting Metabolic Vulnerabilities In Solid Tumors Treated With ABL Kinase Allosteric Inhibitors(2021) Hattaway Luttman, JillianMetastases are common and devastating complications linked to ~90% of cancer deaths. Therapy-resistance is a major challenge for the treatment of cancer cell metastasis as metastatic cells metabolically rewire to survive cytotoxic therapies and adapt to new environments. Understanding and effectively targeting these metabolic changes opens an entirely new therapeutic avenue for combating cancer by defining cancer-related metabolic vulnerabilities. Using a CRISPR/Cas9 loss-of-function screen and RNA-sequencing analysis, the studies presented herein identify two metabolic vulnerabilities that arise following ABL allosteric inhibitor treatment to target metastatic and therapy-resistant cancer cells. First, we identify a novel combination therapy of ABL kinase allosteric inhibitors with lipophilic statins that impairs growth of clinically relevant therapy-resistant and brain metastatic lung cancer cells in vitro and in in vivo using mouse models. We found that ABL allosteric inhibitors impair mitochondria function without altering glycolytic capacity, leading to sensitization to statin therapeutics, and enhanced synergy to promote cancer cell death by combination therapy. Further, we found that ABL inhibitors are sensitized to statins due to the ability of statin therapeutics to inhibit the isoprenoid pathway, specifically protein geranylgeranylation. These results reveal a potential striking clinical benefit as synergy was not noted upon combination with standard of care therapeutics, gefitinib and docetaxel, and identify a new treatment strategy for patients refractory to first-line therapeutics or with metastases to difficult to treat organs like the brain. We have also characterized a novel ABL signaling axis as ABL inhibition was shown to deplete SLC7A11 protein levels in cancer cells. SLC7A11 is the catalytic subunit of system xCT and enables cystine import for cell detoxification and concomitant glutamate export. By depleting cancer cells of SLC7A11, cell detoxification processes are limited and excretion of toxic glutamate levels into the tumor microenvironment decrease. These data suggest that ABL regulation of this pathway could extend survival and relieve harmful symptoms in patients experiencing primary and secondary metastatic tumors. Collectively, our findings reveal metabolic vulnerabilities that can be targeted in cancer cells through treatment with ABL allosteric inhibitors, leading to improved patient survival and quality of life.
Item Open Access Exploring the Role of Mitochondrial Bioenergetics and Metabolism in Heart Failure(2020) Davidson, Michael ThomasHeart failure is a worldwide public health problem with substantial clinical burden and economic costs. In the progression into failure, the heart undergoes dramatic alterations in mitochondrial fuel metabolism and bioenergetics. As such, there is considerable interest in the delineation of regulatory events involved in the metabolic dysfunction of heart failure. Previous collaborative work identified three metabolic signatures associated with early stage heart failure: 1) accumulation of acylcarnitine metabolites; 2) mitochondrial hyperacetylation; and 3) elevated ketone catabolism. The goal of this dissertation was to explore the role of these metabolic signatures in the pathogenesis of heart failure.
Tissue accumulation of acylcarnitine metabolites is characteristic of mitochondrial dysfunction and indicative of incomplete β-oxidation. This occurs when a large portion of the fatty acids (i.e., acyl groups) within the mitochondria are not fully catabolized and the resulting intermediates are transferred to carnitine esters, enabling the traversal of biological membranes and departure from the mitochondrial matrix.
Nϵ-acetylation in the mitochondrial matrix is a non-enzymatic, post-translational modification (PTM) that spontaneously arises from the relatively basic pH and abundance of acetyl-CoA. Accumulation of this PTM has been observed in other tissues and disease states with evidence suggesting it impairs mitochondrial metabolism and causes dysfunction. However, convincing studies are lacking to establish a direct causal connection between dysfunction and acetylation. To address this shortcoming, a novel assay platform for the comprehensive assessment of mitochondrial bioenergetic transduction was developed and validated. Next, we generated and validated a novel mouse model of cardiac mitochondrial hyperacetylation and utilized the bioenergetic assay platform to test the hypothesis that it causes metabolic perturbations. Surprisingly, these hyperacetylated mitochondria exhibited almost no deficits in mitochondrial oxidative metabolism. To determine if hyperacetylation causes mitochondrial dysfunction in vivo under pathologic stimuli, the mouse model and littermate controls were subjected to transaortic constriction, a surgical method to induce pressure-overload heart failure. The hyperacetylated animals did not exhibit enhanced sensitivity toward cardiac dysfunction relative controls. With these results, we concluded that mitochondrial hyperacetylation does not contribute to the pathogenesis of heart failure.
Elevated ketone catabolism was observed in early stage failing hearts. Through a series of murine and canine heart failure models, ketone catabolism was shown to be adaptive in response to pathological stress. Additionally, the mitochondrial bioenergetic assay platform was applied to cardiac mitochondria under substrate limited-conditions. These results indicate that ketone catabolism improves bioenergetic efficiency under constraints which mimic the failing heart. With these results, we conclude ketone catabolism is an important metabolic defense in response to the dysfunction of the failing heart.
Item Open Access Fibroblast Growth Factor 13 Regulates Thermogenesis and Metabolism(2019) Sinden, Daniel StephenThe non-secreted fibroblast growth factor (FGF) homologous factor (FHF) FGF13 is a noncanonical FGF with identified roles in neuronal development, pain sensation, and cardiac physiology, but recent reports suggest broader roles. The in vivo functions of FGF13 have not been widely studied. In this study, we have generated a global heterozygous Fgf13 knockout mouse model. In these animals, we observed hyperactivity and accompanying reduced core body temperature in mice housed at 22 °C. In mice housed at 30 °C (thermoneutrality) we observed development of a pronounced obesity. Defects in thermogenesis and metabolism were found to be due to impaired central nervous system regulation of sympathetic activation of brown fat. Neuronal and hypothalamic specific ablation of Fgf13 recapitulated weight gain at 30 °C. In global heterozygous animals, norepinephrine turnover in brown fat was reduced at both housing temperatures, while direct activation of brown fat by a β3 agonist showed an intact response. Further, we found that FGF13 is a direct regulator of NaV1.7, a hypothalamic Na+ channel associated with regulation of body weight. Our data expand the physiologic roles for FGF13, and enhance the understanding of the multifunctional FHFs.
Item Open Access Genomic Basis for a Developmental Life History Switch in the Sea Urchin Heliocidaris erythrogramma(2021) Davidson, Phillip LukeLecithotrophic (non-feeding) larval development has independently evolved numerous times in marine invertebrates from an ancestral, planktotrophic (feeding) larval state. The evolution of this developmental mode in a species is accompanied by dramatic changes in ecology and development, including lower fecundity, higher maternal investment per offspring, changes in egg composition, alteration of embryonic fate specification, morphologically simple larvae, and reduced time to metamorphosis. Thus, the evolutionary switch between lecithotrophy and planktotrophy serves as an exemplary system for investigating the effect of changing ecological pressures on the evolution of novel developmental phenotypes. The sea urchin genus Heliocidaris represents one of the best studied examples of this switch, in which H. erythrogramma evolved lecithotrophy around five million years ago. Over the past several decades, previous work has documented phenotypes distinguishing development of this species from the ancestral, planktotrophic condition. These phenotypes range from increased sperm size and hypertrophy of lipid deposition in the egg, to changes in embryonic axis determination, delayed blastomere specification, and alterations to spatial and temporal expression of key developmental network genes. Although much is known about what phenotypes are associated with the evolution of lecithotrophy in this species, much less is known of the regulatory mechanisms for how these changes arose in the first place. This gap in knowledge is the subject of my thesis: to gain a better understanding of the genomic and molecular basis for the evolution of lecithotrophy in H. erythrogramma. To accomplish this, I carried out a set of physiological and genomic comparisons between H. erythrogramma, a closely-related planktotrophic congener H. tuberculata, and a distantly-related planktotroph Lytechinus variegatus in order to identify specific molecules and genomic loci underlying lecithotrophic development. In Chapter One, I analyzed lipid and protein content of eggs and larvae from these three species using mass spectrometry to characterize metabolic differences in egg provisioning and embryogenesis in H. erythrogramma. In Chapter 2, I present a chromosome-level assembly of L. variegatus, highlighting a genome assembly and annotation method that will be applied to the two Heliocidaris species and the utility of a high-quality genome assembly for functional genomic analysis. In Chapter 3, I compare the genome assemblies of H. erythrogramma and H tuberculata to show that a conserved developmental network controlling sea urchin development has been dramatically modified in H. erythrogramma through genic and non-coding modifications. In Chapter 4, I compare the chromatin landscapes of these three species through development using ATAC-seq to access how cis-regulatory mechanisms have evolved during the acquisition of lecithotrophic development. From this work, I found that the enormous lipid provisioning of H. erythrogramma eggs is composed primarily of diacylglycerol ether lipids and that these lipids are not metabolized for pre-metamorphic development, but instead provisioned to promote post-metamorphic survivorship of juvenile individuals. Instead, upregulated glycolysis proteins suggest this pathway may be driving rapid pre-metamorphic development. Comparative genomic analyses demonstrate positive selection and changes to chromatin accessibility have modified the regulatory genome of H. erythrogramma, especially near developmental network genes, and that these changes are associated with temporal and spatial differences in embryonic gene expression. Furthermore, the Pmar1 transcription factor family has likely lost its ancestral function in specifying the primary mesenchyme lineage in this species, a cell type responsible for larval skeletal development and patterning of the embryo. Finally, development has one of the largest effects on changes in chromatin accessibility in each species, but particularly near developmental genes, embryonic chromatin dynamics is highly associated with the life history strategy of each species. Future work identifying examples of convergent or novel pathways driving evolution of lecithotrophy in other echinoids will provide valuable insight into general principles governing how derived developmental phenotypes can evolve at short evolutionary timescales.
Item Open Access Glucose metabolism and p53 in leukemia(2011) Mason, Emily FergusonHealthy cells require input from growth factor signaling pathways to maintain cell metabolism and survival. Growth factor deprivation induces a loss of glucose metabolism that contributes to cell death in this context, and we have previously shown that maintenance of glycolysis after growth factor deprivation suppresses the activation of p53 and the induction of the pro-apoptotic protein Puma to prevent cell death. However, it has remained unclear how cell metabolism regulates p53 activation and whether this increased glycolysis promotes cell survival in the face of additional types of cell stress. To examine these questions, we have utilized a system in which stable overexpression of the glucose transporter Glut1 and hexokinase 1 in hematopoietic cells drives growth-factor independent glycolysis. This system allows us to examine the effects of glucose metabolism in the absence of other signaling events activated downstream of growth factor receptors. Here, we demonstrate that elevated glucose metabolism, characteristic of cancer cells, can suppress PKCδ-dependent p53 activation to maintain cell survival after growth factor withdrawal. In contrast, DNA damage-induced p53 activation was PKCδ-independent and was not metabolically sensitive. Both stresses required p53 serine 18 phosphorylation for maximal activity but led to unique patterns of p53 target gene expression, demonstrating distinct activation and response pathways for p53 that were differentially regulated by metabolism.
Unlike the growth factor-dependence of normal cells, cancer cells can maintain growth factor-independent glycolysis and survival and often demonstrate dramatically increased rates of glucose uptake and glycolysis, in part to meet the metabolic demands associated with cell proliferation. Given the ability of elevated glucose metabolism to suppress p53 activity in the context of metabolic stress, we examined the effect of increased glucose uptake on leukemogenesis using a mAkt-driven model of leukemia and adoptive transfer experiments. We show here that elevated glucose uptake promoted leukemogenesis in vivo, perhaps through suppression of p53 transcriptional activity. During the process of leukemogenesis, cancerous cells can acquire growth factor independent control over metabolism and survival through expression of oncogenic kinases, such as BCR-Abl. While targeted kinase inhibition can promote cancer cell death, therapeutic resistance develops frequently and further mechanistic understanding regarding these therapies is needed. Kinase inhibition targets the necessary survival signals within cancerous cells and may activate similar cell death pathways to those initiated by growth factor deprivation. As we have demonstrated that loss of metabolism promotes cell death after growth factor withdrawal, we investigated whether cell metabolism played a role in the induction of apoptosis after treatment of BCR-Abl-expressing cells with the tyrosine kinase inhibitor imatinib. Consistent with oncogenic kinases acting to replace growth factors, treatment of BCR-Abl-expressing cells with imatinib led to reduced metabolism and p53- and Puma-dependent cell death. Accordingly, maintenance of glucose uptake inhibited p53 activation and promoted imatinib resistance, while inhibition of glycolysis enhanced imatinib sensitivity in BCR-Abl-expressing cells with wild type p53 but had little effect on p53 null cells. Together, these data demonstrate that distinct pathways regulate p53 after DNA damage and metabolic stress and that inhibition of glucose metabolism may enhance the efficacy of and overcome resistance to targeted molecular cancer therapies.
Item Open Access Glutaminase Modulates T Cell Metabolism and Function in Inflammation and Cancer(2018) Johnson, Marc ODuring the immune response, helper T cells must proliferate and upregulate key metabolic programs including glucose and glutamine uptake. Metabolic reprogramming is imperative for appropriate T cell responses, as inhibition of glucose or glutamine uptake hinders T cell effector responses. Glutamine and glutaminolysis use in cancer cells has partially been explored. However, the role of glutamine and its downstream metabolites is incomplete and unclear in T cells. The first step of glutamine metabolism is conversion to glutamate via the hydrolase enzyme glutaminase (GLS). To target glutaminolysis, two different methods were employed: 1) genetic knockout of GLS using a CRE-recombinase system specific for CD4/CD8 T cells, and 2) pharmacological inhibition of GLS via the potent and specific small molecular CB839. These two models of glutaminase insufficiency were used as a tool to target glutamine metabolism during T cell activation and differentiation both in vitro and in vivo.
GLS-deficient T cells had decreased activation at early time points compared to control. Over several days, these GLS-deficient T cells differentiated preferentially to Th1-like effector cells. This was reliant on increased glucose carbons incorporating into Tri-Carboxylic Acid (TCA) metabolites. This increased effector response in vitro occurred in both CD4+ T helper cells and CD8+ cells (Cytotoxic lymphocytes, or CTLs). Differentiation of CD4+ T cells to Th1 or Th17 subsets showed decreased Th17 differentiation and cytokine production, while Th1 effector responses were increased. This increased Th1 function was dependent on IL-2 signaling and mTORC1, as reducing IL-2 or inhibiting mTORC1 with rapamycin prevented GLS inhibition-induced Th1 effector function. Th17 cells, meanwhile, were inhibited by changes in reactive oxygen species, and recovery of Th17 function was achieved with n-acetylcysteine treatment.
T cells lacking GLS were unable to induce inflammation in a mouse model of Graft vs Host disease, an inflammatory bowel disease model, or in an airway inflammatory model. Importantly, Chimeric Antigen Receptor (CAR) T cells made from GLS knockout cells were unable to maintain B cell aplasia in recipient mice. Contrary to this, temporary inhibition of GLS via small-molecule inhibition increased B cell killing in vitro and enhanced T cell persistence in both the B cell aplasia and in a vaccinia virus recall response. These results indicate a balance, where permanent deficiency of GLS is detrimental to T cell responses, but acute inhibition can actually promote T effector responses and survival. Overall, this work aims to understand how perturbations in glutamine metabolism in T cells affects differentiation and function and the role of glutaminolysis and improve therapies for inflammatory disease and cancer.
Item Open Access Harnessing Optical Imaging for Assessing Metabolic Reprogramming in Breast Cancer(2020) Madonna, Megan CathleenAccording to the World Health Organization, there were over 2 million new breast cancer cases in 2018. This number is projected to steadily increase year after year. American Cancer Society projections for 2020 list the breast as the leading cancer site for new cancer cases in females, estimating breast cancer to represent 30% of all new cases and 15% of cancer-related deaths.
A leading cause of breast cancer deaths is due to tumor recurrence following therapy. These tumors can recur years, sometimes decades, after treatment from reservoirs of residual cells that persist in a dormant state. Conversely, the absence of residual invasive disease following adjuvant therapy constitutes pathological complete response (pCR) and is positively associated with long-term relapse-free survival. This risk for recurrence is higher for women with human epidermal growth factor receptor 2 (Her2+) breast cancer or triple-negative breast cancer (TNBC). Approximately 50-70% of Her2+ patients and 40-55% of TNBC patients who undergo standard therapy achieve pCR; however, in the remaining patients, only a partial response occurs, leaving residual disease and an increased risk of relapse.
To mitigate the cancer burden, years of research have focused on several common biological capabilities of cancer, deemed the Hallmarks of Cancer, including sustained proliferation, genome mutations, replicative immortality, resistance to cell death, and a deregulated metabolism. Several recent studies have further reported that this last hallmark, metabolism, may be vital to understanding the underlying behavior of dormant and recurrent tumors. Once understood, these changes in metabolic pathways, referred to as metabolic reprogramming, can be leveraged as vulnerabilities and allow for the development of strategies to eliminate residual disease or prevent residual tumor cells’ subsequent reactivation into full recurrence.
For nearly 100 years, increased aerobic glycolysis has been considered a feature of rapidly proliferating primary tumors. This occurrence, where cells continue to use the metabolic pathway where glucose is converted to lactic acid to release its stored energy and produce adenosine triphosphate (ATP) despite the presence of oxygen, has been termed the Warburg Effect. Because of this, physicians frequently use nuclear medicine directly imaging glucose uptake, fluorodeoxyglucose (FDG) Positron Emission Tomography (PET) imaging, for the diagnosis and staging of cancer. In addition to glycolysis, mitochondrial metabolism through oxidative phosphorylation has grown in recognition as an additional energy source for cancer cells. In mitochondrial metabolism, the tricarboxylic acid (TCA) cycle generates energy carriers to be used in the electron transport chain. Here, the mitochondrial membrane potential provides a gradient to produce large amounts of ATP. Additionally, the TCA cycle can rely on sources of carbon besides glucose alone. A steadily growing consensus points to other energetic sources, such as glutamine, amino acids, and lipids, that are key to survival, especially following environmental stress, treatment, or before migration and metastasis.
Though metabolic reprogramming underpins aspects of tumor dormancy and recurrence, currently, there are no techniques available to provide a systems-level approach to investigate the major axes of metabolism. Several techniques that offer insights into cellular metabolism exist, such as the Seahorse assay, metabolomics, and FDG-PET imaging. They, however, are limited to in vitro model systems, single-time point analyses of in vivo model systems, or single-endpoint analysis of in vivo model systems, respectively. Further, neither the Seahorse assay nor metabolomics can capture information about both the tumor and its native microenvironment. Therefore, there is an unmet need for a method to study metabolism at a spatial resolution that can elucidate the metabolic modulation of residual cell populations longitudinally and across in vitro and in vivo models.
Optical imaging is well-suited to address this gap in technologies owing to its ability to measure multiple metabolic endpoints non-destructively and repeatedly. The Center for Global Women’s Health Technologies has developed protocols for the use of two optical probes 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethylrhodamine, ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, in preclinical cancer models. These endpoints are superior to imaging of the endogenous fluorescence of NADH and FAD (referred to as the redox ratio) by providing a direct measure of a substrate (glucose uptake) and metabolic output (mitochondrial metabolism). This optical, metabolic imaging approach fills a critical gap that exists between in vitro studies on single cells (Seahorse Extracellular Flux Assay) and whole-body imaging (FDG-PET imaging) and is complementary to metabolomics and immunohistochemistry (IHC) with endpoints measuring the major axes of metabolism.
The work described here details an innovative platform to image changes in the metabolism of primary tumors, residual disease, and recurrent tumors using a Her2+ genetically engineered mouse model. This model exhibits key features of dormancy and mimics sustained use of targeted therapy to facilitate understanding of tumor biology and function, assess recurrence risk, and design therapies to mitigate residual disease and recurrence altogether. Imaging at a cellular level resolution will not only document acute metabolic changes following Her2 downregulation but also allow for metabolic imaging of dormant cell populations that are typically too small to study in human patients, typically referred to as no evidence of disease (NED) in humans. This platform will push metabolic studies of tumor dormancy further.
Three specific aims were proposed towards this ultimate goal to develop a multiparametric platform to characterize the metabolic reprogramming of preclinical cancer models.
Aim 1 establishes the functional flexibility of the fluorescent glucose analog 2-NBDG to measure glycolytic demand and the fluorescent cation TMRE to measure mitochondrial membrane potential to report on the metabolic changes that occur throughout tumor progression, dormancy, and recurrence. Using a genetically engineered mouse-derived three-dimensional in vitro mammosphere model allowed for metabolic endpoints to be captured across key time points. Doxycycline (dox) addition and withdrawal modulates expression of Her2, which is overexpressed in primary and re-activated mammospheres, and downregulated in regressing and dormant mammospheres. The mammospheres were characterized using immunofluorescence to confirm phenotype. Ki67 expression was high in primary and re-activated mammospheres, confirming a proliferative phenotype typical of both primary and recurrent disease presented in the clinic. On the other hand, short-term dox withdrawal resulted in increased cleaved caspase 3 (CC3) expression, confirming apoptosis due to Her2 downregulation. Finally, both Ki67 and CC3 expression were negative in dormant mammospheres, demonstrating a viable, but non-proliferative, steady-state phenotype.
Metabolic imaging revealed unique metabolic phenotypes across the tumor development stages that were consistent with the gold standard assays. While primary mammospheres, overexpressing Her2, maintained increased glucose uptake (“Warburg effect”), after Her2 downregulation, regressing and residual disease mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, re-eliciting growth. These findings highlight the importance of imaging metabolic adaptations to gain insight into residual and recurrent disease’s fundamental behaviors.
This work paved the way for similar studies in vivo using a mammary window chamber with the ultimate goal of informing the potential impact of metabolically-targeted therapies on tumor dormancy and recurrence.
In Aim 2, 2-NBDG and TMRE imaging was applied to in vivo mammary tumors as they transitioned from primary tumors, through regression and dormancy, to regrowth as recurrent tumors. Two tumor models varying in periods of dormancy (termed slow recurring and fast recurring tumors) were selected to characterize the importance of either axis of metabolism in the context of recurrent disease. When comparing the glucose demand and mitochondrial membrane potential levels between slow and fast recurring tumors, both sets of primary tumors behaved similarly to the primary mammosphere cultures: increased 2-NBDG indicating highly glycolytic tumors with low TMRE indicating little mitochondrial activity. Following acute Her2 downregulation, there was an increase of mitochondrial activity that remained relatively constant through regression, dormancy, and recurrence for both tumor types. However, glucose uptake varied between the two tumor types following Her2 downregulation. The mice bearing slow-recurring tumors showed a resurgence of glucose uptake during recurrence; conversely, the mice bearing fast-recurring tumors maintained decreased glucose levels continually following Her2 downregulation. Because the fast-recurring tumors did not have a meaningful change in glucose uptake during recurrence, it was hypothesized that the fast-recurring tumors might have reprogrammed to use fatty acids as a fuel source. Indeed, inhibiting fatty acid oxidation in these tumors resulted in increased glucose uptake during regression. Additionally, following this acute change in metabolism due to the inhibition of fatty acid oxidation, the tumor’s dormancy period prior to recurrence was prolonged, pointing to lipids as a crucial fuel source for residual disease and recurrence in aggressive breast cancer.
Aim 2 showed the importance of lipid metabolism in residual disease and recurrence. Additionally, other groups have also shown increased reliance on fatty acid oxidation in breast cancer residual disease following oncogene downregulation. Thus, Aim 3 established a method of visualizing long-chain fatty acid uptake in breast cancer murine models. Until now, the ability to monitor such uptake has been limited to in vitro and ex vivo approaches. Here, an imaging strategy that combines a fluorescently labeled palmitate molecule, Bodipy FL c16, and intravital, optical imaging was developed to measure exogenous fatty acid uptake. Because the palmitate’s 16th carbon is fluorescently labeled, immediate degradation of the Bodipy dye during fatty acid oxidation (β-oxidation) is prevented, allowing for fatty acid to be visualized through fluorescence imaging.
This technique was validated in two breast cancer models: a MYC-overexpressing transgenic triple-negative breast cancer (TNBC) model, previously reported to dramatically upregulate fatty acid oxidation intermediates, and the murine model of the 4T1 family, a group of sibling tumor lines with a reported wide range of metabolic phenotypes.
Using a genetically engineered mouse-derived xenograft allowed for fatty acid uptake levels to be captured during MYC-overexpression and following oncogene downregulation. Similar to the previously described genetically engineered model, this model used doxycycline addition and withdrawal to modulate MYC expression.
Through in vivo Bodipy FL c16 imaging, fatty acid uptake was found to be increased in MYC-high tumors. This model showcased two critically needed features for clinically relevant study of fatty acid uptake: 1) longitudinal metabolite tracking in a single animal shown through intra-animal decreases in fatty acid uptake following MYC-downregulation; and 2) providing a link between oncogene expression, which can be modulated therapeutically, and metabolic endpoints. This decreased uptake is indicative of a less aggressive state and correlates with a visible reduction in tumor volume. Additionally, this method found an increased fatty acid uptake in tumors with high metastatic potential, as well as the ability of the system to monitor inhibition efficacy, potentially allowing for therapeutic pharmaceutical testing of drug efficacy.
This fast and dynamic approach to image fatty acid uptake in vivo is a tool relevant to study tumor metabolic reprogramming or the effectiveness of drugs targeting lipid metabolism.
Targeting a tumor’s metabolic dependencies is a clinically actionable therapeutic approach, but identifying subtypes of tumors that are likely to respond remains difficult. The work presented here indicates that an optical platform to image 2-NBDG, TMRE, and Bodipy FL c16 longitudinally is well suited to characterize breast cancer residual disease and recurrence’s critical metabolic features and to pinpoint metabolic vulnerabilities for potential treatments. While the primary goal was to develop an imaging strategy for the unprecedented assessment of residual and recurrent disease at high resolution in in vitro and in vivo models, this innovation also fits within the broader framework of existing metabolic assessment techniques and provides a systematic way to connect in vitro studies to whole-body imaging within the context of preclinical pharmacology research.
Future work will focus on establishing a combined imaging strategy for simultaneous imaging of all three endpoints, transitioning imaging to a hand-held microscope for wide-spread adoption and rapid metabolic phenotyping of clinical samples, and integrating optical spectroscopy with this imaging platform to track the long-term effects therapy has on an individual tumor’s metabolism. The third will enable the ability to retrospectively look for changes in primary and regressing phenotypes that might foreshadow dormant behavior or the risk of early recurrence.
Item Open Access Harnessing T Cell Generation and Metabolism to Modulate T Cell Recovery Following Radiation Exposure and Bone Marrow Transplantation(2022) Zou, YujingTotal body irradiation (TBI) causes profound suppression of hematopoiesis and T cell depletion, increasing chances of morbidity associated with opportunistic infections in the lymphopenic condition. Currently, therapeutic options for improving recovery of the T cell compartment following radiation exposure are not available. Although mouse and nonhuman primate studies have demonstrated prolonged effects of TBI on T cell reconstitution, there is a lack of understanding in the kinetics and metabolic signatures of radioresistant T cells actively undergoing homeostatic proliferation. Furthermore, whether kinetics of systemic T cell recovery recapitulates T cell recovery in circulation remains unknown. In the current study, we performed comprehensive immunophenotyping and single-cell sequencing analyses of radioresistant T cells, as well as imaging of T cell recovery in vivo, to determine preferentially upregulated pathways during T cell recovery. We identified T cell populations unique to TBI treatment that upregulate components essential to support oxidative phosphorylation, a mitochondria-dependent metabolic process. We further investigated mechanisms of recovery in donor T cells following TBI exposure in the bone marrow transplant setting. We demonstrated that recovery of alloreactive donor T cells was highly dependent on aerobic glycolysis, which can be manipulated to reduce graft-versus-host-disease and preserve the functional recovery of non-alloreactive donor T cells. We then examined the effect of NT-I7, a long-acting recombinant human IL-7, in mediating T cell reconstitution due to its role in integrating metabolic requirements with pathways critical for T cell survival and growth. We found that NT-I7 led to accelerated T cell recovery following TBI through both thymic-dependent and independent pathways. More importantly, NT-I7 promoted functional T cell recovery. Taken together, these findings reveal unique kinetics and mechanisms of T cell recovery in response to radiation. The study also identified NT-I7 as a potential therapeutic treatment during T cell lymphopenia by supporting critical mechanisms utilized in T cell recovery.
Item Open Access Heritability estimates of endophenotypes of long and health life: the Long Life Family Study.(J Gerontol A Biol Sci Med Sci, 2010-12) Matteini, Amy M; Fallin, M Daniele; Kammerer, Candace M; Schupf, Nicole; Yashin, Anatoli I; Christensen, Kaare; Arbeev, Konstantin G; Barr, Graham; Mayeux, Richard; Newman, Anne B; Walston, Jeremy DBACKGROUND: Identification of gene variants that contribute to exceptional survival may provide critical biologic information that informs optimal health across the life span. METHODS: As part of phenotype development efforts for the Long Life Family Study, endophenotypes that represent exceptional survival were identified and heritability estimates were calculated. Principal components (PCs) analysis was carried out using 28 physiologic measurements from five trait domains (cardiovascular, cognition, physical function, pulmonary, and metabolic). RESULTS: The five most dominant PCs accounted for 50% of underlying trait variance. The first PC (PC1), which consisted primarily of poor pulmonary and physical function, represented 14.3% of the total variance and had an estimated heritability of 39%. PC2 consisted of measures of good metabolic and cardiovascular function with an estimated heritability of 27%. PC3 was made up of cognitive measures (h(2) = 36%). PC4 and PC5 contained measures of blood pressure and cholesterol, respectively (h(2) = 25% and 16%). CONCLUSIONS: These PCs analysis-derived endophenotypes may be used in genetic association studies to help identify underlying genetic mechanisms that drive exceptional survival in this and other populations.Item Restricted Homeostatic imbalance of purine catabolism in first-episode neuroleptic-naïve patients with schizophrenia.(PLoS One, 2010-03-03) Yao, Jeffrey K; Dougherty, George G; Reddy, Ravinder D; Keshavan, Matcheri S; Montrose, Debra M; Matson, Wayne R; McEvoy, Joseph; Kaddurah-Daouk, RimaBACKGROUND: Purine catabolism may be an unappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. Accumulating evidence suggests a pivotal role of oxidative stress in schizophrenia pathology. METHODOLOGY/PRINCIPAL FINDINGS: Using high-pressure liquid chromatography coupled with a coulometric multi-electrode array system, we compared 6 purine metabolites simultaneously in plasma between first-episode neuroleptic-naïve patients with schizophrenia (FENNS, n = 25) and healthy controls (HC, n = 30), as well as between FENNS at baseline (BL) and 4 weeks (4w) after antipsychotic treatment. Significantly higher levels of xanthosine (Xant) and lower levels of guanine (G) were seen in both patient groups compared to HC subjects. Moreover, the ratios of G/guanosine (Gr), uric acid (UA)/Gr, and UA/Xant were significantly lower, whereas the ratio of Xant/G was significantly higher in FENNS-BL than in HC. Such changes remained in FENNS-4w with exception that the ratio of UA/Gr was normalized. All 3 groups had significant correlations between G and UA, and Xan and hypoxanthine (Hx). By contrast, correlations of UA with each of Xan and Hx, and the correlation of Xan with Gr were all quite significant for the HC but not for the FENNS. Finally, correlations of Gr with each of UA and G were significant for both HC and FENNS-BL but not for the FENNS-4w. CONCLUSIONS/SIGNIFICANCE: During purine catabolism, both conversions of Gr to G and of Xant to Xan are reversible. Decreased ratios of product to precursor suggested a shift favorable to Xant production from Xan, resulting in decreased UA levels in the FENNS. Specifically, the reduced UA/Gr ratio was nearly normalized after 4 weeks of antipsychotic treatment. In addition, there are tightly correlated precursor and product relationships within purine pathways; although some of these correlations persist across disease or medication status, others appear to be lost among FENNS. Taken together, these results suggest that the potential for steady formation of antioxidant UA from purine catabolism is altered early in the course of illness.Item Open Access Identification of Molecular Determinants of Cellular Senescence in Cancer and Aging(2018) Yuan, LifengCellular senescence is a fundamental cell fate playing significant and complex roles during tumorigenesis and natural aging process. However, the molecular determinants distinguishing senescence from other temporary and permanent cell-cycle arrest states such as quiescence and post-mitotic state and the specified mechanisms underlying cell-fate decisions towards senescence versus cell death in response to cellular stress stimuli remain less understood. In our studies, we aimed to employ multi-omics approaches to deepen our understanding of cellular senescence, in particular, regarding the specific molecular determinants distinguishing cellular senescence from other non-dividing cell fates.
Notably, one of the most prominent features of cellular senescence differing from other non-dividing cell fates is the increased expression of senescence-associated beta-galactosidase. Because 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG) is known as the substrate catalyzed by beta-galactosidase for producing a green fluorescent product, we applied this compound to the cells undergoing G1 cell-cycle arrest (a mixture of senescent and quiescent cells). Employing fluorescence-activated cell sorting, we separated and collected senescent and quiescent cell populations based on green fluorescence intensity. As cellular senescence is more than just the non-dividing cell fate, we therefore systematically compared the gene expression between senescence and quiescence to provide insights into the specific features underlying senescence programming beyond cell cycle arrest. Following this strategy for the comparative gene expression analysis, we identified and characterized several genes critically involved in the program of cellular senescence, and one of the major findings was to identify IMMP2L, a nuclear-encoded mitochondrial intermembrane peptidase, can act as a molecular switch for determining the cell fates of healthy living, cell death, and senescence.
Inhibiting IMMP2L signaling through either the suicidal protease inhibitor SERPINB4 or transcriptional downregulation was sufficient to initiate cellular senescence by reprogramming the mitochondria functionality. Employing proteomics, we identified at least two mitochondrial target proteins processed by IMMP2L, including metabolic enzyme GPD2 and cell death regulator/electron transport chain complex I component AIF. Functional study suggests that, in healthy cells, the IMMP2L-GPD2 axis catalyzes redox reactions to produce phospholipid precursor Glycerol 3-phosphate; while under oxidative stress, IMMP2L cleaves AIF into its truncated pro-apoptotic form leading to cell death initiation to remove cells with irreparable damage. For cells programmed to senesce, the IMMP2L-GPD2 axis is switched off to block phospholipid biosynthesis leading to reduced availability of membrane building blocks for cell growth together with the disruption of mitochondrial localization of certain phospholipid-binding kinases, such as protein kinase C-δ (PKC-δ) and its downstream signaling. These alterations in mitochondria-associated metabolism and signaling network promote entry into a senescent state featuring high levels of reactive oxygen species (ROS). Simultaneously, blockage of pro-apoptotic AIF generation, which is due to the loss of IMMP2L, ensures the viability of senescent cells under ROS-mediated oxidative stress. Taken together, we have mechanistically uncovered IMMP2L-mediated signaling as a key regulatory pathway in the control of fates of healthy, apoptotic, and senescent cells.
In the physiological conditions, we observed that IMMP2L is downregulated in the muscle tissues and the blood samples of geriatric groups compared to that from young cohorts. Besides, centenarians display better genomic integrity at the IMMP2L locus when compared with the general population. Taken together, it suggests IMMP2L could also be an important player associated with the natural aging process.
Item Open Access Investigating the Intrinsic and Extrinsic Drivers of Primate Heterothermy(2016) Faherty, Sheena LeeSeasonal heterothermy—an orchestrated set of extreme physiological responses—is directly responsible for the over-winter survival of many mammalian groups living in seasonal environments. Historically, it was thought that the use of seasonal heterothermy (i.e. daily torpor and hibernation) was restricted to cold-adapted species; it is now known that such thermoregulatory strategies are used by more species than previously appreciated, including many tropical species. The dwarf and mouse lemurs (family Cheirogaleidae) are among the few primates known to use seasonal heterothermy to avoid Madagascar’s harsh and unpredictable environments. These primates provide an ideal study system for investigating a common mechanism of mammalian seasonal heterothermy. The overarching theme of this dissertation is to understand both the intrinsic and extrinsic drivers of heterothermy in three species of the family Cheirogaleidae. By using transcriptome sequencing to characterize gene expression in both captive and natural settings, we identify unique patterns of differential gene expression that are correlated with extreme changes in physiology in two species of dwarf lemurs: C. medius under captive conditions at the Duke Lemur Center and C. crossleyi studied under field conditions in Madagascar. Genes that are differentially expressed appear to be critical for maintaining the health of these animals when they undergo prolonged periods of metabolic depression concurrent with the hibernation phenotype. Further, a comparative analysis of previously studied mammalian heterotherms identifies shared genetic mechanisms underlying the hibernation phenotype across the phylogeny of mammals. Lastly, conducting a diet manipulation study with a captive colony of mouse lemurs (Microcebus murinus) at the Duke Lemur Center, we investigated the degree to which dietary effects influence torpor patterns. We find that tropical primate heterotherms may be exempt from the traditional paradigms governing cold-adapted heterothermy, having evolved different dietary strategies to tolerate circadian changes in body temperature.