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Table of Contents forMetabolic Genomics & Pharmaceutics, Vol. I
Chapter 1: Metabolic Pathways
Chapter 2: Lipid Metabolism
Chapter 3: Cell Signaling
Chapter 4: Protein Synthesis and Degradation
Chapter 5: Sub-cellular Structure
Chapter 6: Proteomics
Chapter 7: Metabolomics
Chapter 8: Impairments in Pathological States: Endocrine Disorders; Stress
Hypermetabolism and Cancer
Chapter 9: Genomic Expression in Health and Disease
Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’
Writer and Curator: Larry H. Bernstein, MD, FCAP
(Note that each portion of the discussion is followed by a reference)
It is now a time to pause after almost a century of a biological scientific discoveries that have transformed the practice of medicine and impacted the lives of several generations of young minds determined to probe the limits of our knowledge. In the century that we have entered into the scientific framework of medicine has brought together a difficult to grasp evolution of the emergence of human existence from wars, famine, droughts, storms, infectious diseases, and insect born pestilence with betterment of human lives, only unevenly divided among societal classes that have existed since time immemorial. In this short time span there have emerged several generations of physicians who have benefited from a far better medical education that their forebears could have known. In this expansive volume on cancer, we follow an incomplete and continuing challenge to understand cancer, a disease that has become associated with longer life spans in developed nations.
While there are significant improvements in the diagnosis and treatment of cancers, there is still a personal as well as locality factor in the occurrence of this group of diseases, which has been viewed incorrectly as a “dedifferentiation” of mature tissue types and the emergence of a cell phenotype that is dependent on glucose, reverts to a cancer “stem cell type” (loss of stemness), loses cell to cell adhesion, loses orderly maturation, and metastasizes to distant sites. At the same time, physician and nurses are stressed in the care of patients by balancing their daily lives and maintaining a perspective.
The conceptual challenge of cancer diagnosis and management has seemed insurmountable, but owes much to the post World War I activities of Otto Heinrich Warburg. It was Warburg who made the observation that cancer cells metabolize glucose by fermentation in much the way Pasteur 60 years earlier observed fermentation of yeast cells. This metabolic phenomenon occurs even in the presence of an oxygen supply, which would provide a huge deficit in ATP production compared with respiration. The cancer cell is “addicted to glucose” and produced lactic acid. Warburg was awarded the Nobel Prize in Medicine for this work in 1931.
In the last 15 years there has been a resurgence of work on the Warburg effect that sheds much new light on the process that was not previously possible, with significant therapeutic implications. In the first place, the metabolic mechanism for the Warburg effect was incomplete even at the beginning of the 21st century. This has been partly rectified with the enlightening elucidation of genome modifications, cellular metabolic regulation, and signaling pathways.
The following developments have become central to furthering our understanding of malignant transformation.
There is usually an identifiable risk factor, such as, H. pylori, or of a chronic inflammatory state, as in the case of Barrett’s esophagus.
There are certain changes in glucose metabolism that have been unquestionably been found in the evolution of this disease. The changes are associated with major changes in metabolic pathways, miRN signaling, and the metabolism geared to synthesis of cells with an impairment of the cell death cycle. In these changes, mitochondrial function is central to both the impaired respiration and the autophagy geared to the synthesis of cancer cells.
The emergence of this cell prototype is characterized by the following, again related to the Warburg effect:
Cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis
The mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis.
Cancer cells tend to express a partially inhibited splice variant of pyruvate kinase (PK-M2), leading to decreased pyruvate production.
The two proteins that mediate pyruvate conversion to lactate and its export, M-type lactate dehydrogenase and the monocarboxylate transporter MCT-4, are commonly upregulated in cancer cells leading to decreased pyruvate oxidation.
The enzymatic step following mitochondrial entry is the conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex. Cancer cells frequently exhibit increased expression of the PDH kinase PDK1, which phosphorylates and inactivates PDH. This PDH regulatory mechanism is required for oncogene induced transformation and reversed in oncogene-induced senescence.
The PDK inhibitor dichloroacetate has shown some clinical efficacy, which correlates with increased pyruvate oxidation. One of the simplest mechanisms to explain decreased mitochondrial pyruvate oxidation in cancer cells, a loss of mitochondrial pyruvate import, has been observed repeatedly over the past 40 years. This process has been impossible to study at a molecular level until recently, however, as the identities of the protein(s) that mediate mitochondrial pyruvate uptake were unknown.
The mitochondrial pyruvate carrier (MPC) as a multimeric complex that is necessary for efficient mitochondrial pyruvate uptake. The MPC contains two distinct proteins, MPC1 and MPC2; the absence of either leads to a loss of mitochondrial pyruvate uptake and utilization in yeast, flies, and mammalian cells.
A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth
In addition to the above, the following study has therapeutic importance:
Glycolysis has become a target of anticancer strategies. Glucose deprivation is sufficient to induce growth inhibition and cell death in cancer cells. The increased glucose transport in cancer cells has been attributed primarily to the upregulation of glucose transporter 1 (Glut1), 1 of the more than 10 glucose transporters that are responsible for basal glucose transport in almost all cell types. Glut1 has not been targeted until very recently due to the lack of potent and selective inhibitors.
First, Glut1 antibodies were shown to inhibit cancer cell growth. Other Glut1 inhibitors and glucose transport inhibitors, such as fasentin and phloretin, were also shown to be effective in reducing cancer cell growth. A group of inhibitors of glucose transporters has been recently identified with IC50 values lower than 20mmol/L for inhibiting cancer cell growth. However, no animal or detailed mechanism studies have been reported with these inhibitors.
Recently, a small molecule named STF-31 was identified that selectively targets the von Hippel-Lindau (VHL) deficient kidney cancer cells. STF-31 inhibits VHL deficient cancer cells by inhibiting Glut1. It was further shown that daily intraperitoneal injection of a soluble analogue of STF-31 effectively reduced the growth of tumors of VHL-deficient cancer cells grafted on nude mice. On the other hand, STF-31 appears to be an inhibitor with a narrow cell target spectrum.
These investigators recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like mechanism. These compounds target Glut1 and are efficacious in vivo as anticancer agents. A novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule.
A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo
Alterations in cellular metabolism are among the most consistent hallmarks of cancer. These investigators have studied the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, M-type lactate dehydrogenase levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.
The results are consistent with a functional connection between alterations in glucose metabolism and mitochondrial physiology in cancer. The data also reflect that the dependency of tumor cells on glucose metabolism is a liability for these cells under limited-oxygen conditions. Interfering with LDH-A activity as a means of blocking pyruvate to lactate conversion could be exploited therapeutically. Because individuals with complete deficiency of LDH-A do not show any symptoms under ordinary circumstances, the genetic data suggest that inhibition of LDH-A activity may represent a relatively nontoxic approach to interfere with tumor growth.
Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance
The widespread clinical use of positron-emission tomography (PET) for the detection of aerobic glycolysis in tumors and recent findings have rekindled interest in Warburg’s theory. Studies on the physiological changes in malignant conversion provided a metabolic signature for the different stages of tumorigenesis; during tumorigenesis, an increase in glucose uptake and lactate production have been detected. The fully transformed state is most dependent on aerobic glycolysis and least dependent on the mitochondrial machinery for ATP synthesis.
Tumors ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumors and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumors leads to enhanced, oxygen-independent glucose usage and a lactate based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualized cancer therapy based on the determination of metabolic changes in tumors that might enable the targeted inhibition of invasion and metastasis.
Other important links between cancer-causing genes and glucose metabolism have been already identified. Activation of the oncogenic kinase Akt has been shown to stimulate glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Such tumor cells have been shown to be dependent on glucose because the ability to induce fatty acid oxidation in response to glucose deprivation is impaired by activated Akt. In addition, AMP-activated protein kinase (AMPK) has been identified as a link between glucose metabolism and the cell cycle, thereby implicating p53 as an essential component of metabolic cell-cycle control.
Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted
The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both contributing toapoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DJm, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.
Cancer progression and its resistance to treatment depend, at least in part, on suppression of apoptosis. Although mitochondria are recognized as regulators of apoptosis, their importance as targets for cancer therapy has not been adequately explored or clinically exploited. In 1930, Warburg suggested that mitochondrial dysfunction in cancer results in a characteristic metabolic phenotype, that is, aerobic glycolysis (Warburg, 1930). Positron emission tomography (PET) imaging has now confirmed that most malignant tumors have increased glucose uptake and metabolism. This bioenergetic feature is a good marker of cancer but has not been therapeutically pursued..
The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolic electrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.
A Mitochondria-K+ Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth
Sebastien Bonnet, Stephen L. Archer, Joan Allalunis-Turner, et al.
Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state. Aim: To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Conclusion: Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-β, a cell transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic antileukemic drugs such as STI571 (Gleevec). Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high efficacy nongenetic novel targets for cancer therapies.
A Metabolic Hypothesis of Cell Growth and Death in Pancreatic Cancer
Laszlo G. Boros, Wai-Nang Paul Lee, and Vay Liang W. Go
Pancreas 2002; 24(1):26–33
Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets.
MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoral heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs.
MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.
Clear cell carcinoma (ccRCC) is the commonest subtype of renal cell carcinoma, accounting for 80% of cases. These tumors are highly resistant to cytotoxic chemotherapy and until recently, systemic treatment options for advanced ccRCC were limited to cytokine based therapies, such as interleukin-2 and interferon-α. Recently, anti-angiogenic drugs and mTOR inhibitors, all targeting the HIF–VEGF axis which is activated in up to 91% of ccRCCs through loss of the VHL tumor suppressor gene [1], have been shown to be effective in metastatic ccRCC [2–5]. Although these drugs increase overall survival to more than 2 years [6], resistance invariably occurs, making the identification of new molecular targets a major clinical need to improve outcomes in patients with metastatic ccRCC.
Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target
Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.
Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and a catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of >90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.
O2 concentrations are significantly reduced in many human cancers compared with the surrounding normal tissue. The median PO2 in breast cancers is 10 mmHg, as compared with65 mmHg in normal breast tissue. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy.
HIF-1 is a transcription factor that consists of an O2 regulated HIF-1a and a constitutively expressed HIF-1b subunit. In well-oxygenated cells, HIF-1a is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and a-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts. Prolylhydroxylated HIF-1a is bound by the von Hippel–Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1a for proteasomal degradation (Figure 1a). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases.
The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 – up or down – results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity.
HIF-1: upstream and downstream of cancer metabolism
Gregg L Semenza
Current Opinion in Genetics & Development 2010, 20:51–56
This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Tony Hunter and Richard Marais
Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1a overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.
Intratumoral hypoxia The majority of locally advanced solid tumors contain regions of reduced oxygen availability. Intratumoral hypoxia results when cells are located too far from a functional blood vessel for diffusion of adequate amounts of O2 as a result of rapid cancer cell proliferation and the formation of blood vessels that are structurally and functionally abnormal. In the most extreme case, O2 concentrations are below those required for survival, resulting in cell death and establishing a selection for cancer cells in which apoptotic pathways are inactivated, anti-apoptotic pathways are activated, or invasion/metastasis pathways that promote escape from the hypoxic microenvironment are activated. This hypoxic adaptation may arise by alterations in gene expression or by mutations in the genome or both and is associated with reduced patient survival.
Hypoxia-inducible factor 1 (HIF-1) The expression of hundreds of genes is altered in each cell exposed to hypoxia. Many of these genes are regulated by HIF-1. HIF-1 is a heterodimer formed by the association of an O2-regulated HIF1a subunit with a constitutively expressed HIF-1b subunit. The structurally and functionally related HIF-2a protein also dimerizes with HIF-1b and regulates an overlapping battery of target genes. Under nonhypoxic conditions, HIF-1a (as well as HIF-2a) is subject to O2-dependent prolyl hydroxylation and this modification is required for binding of the von Hippel–Lindau tumor suppressor protein (VHL), which also binds to Elongin C and thereby recruits a ubiquitin ligase complex that targets HIF-1a for ubiquitination and proteasomal degradation. Under hypoxic conditions, the rate of hydroxylation and ubiquitination declines, resulting in accumulation of HIF-1a. Immunohistochemical analysis of tumor biopsies has revealed high levels of HIF-1a in hypoxic but viable tumor cells surrounding areas of necrosis.
Genetic alterations in cancer cells increase HIF-1 activity In the majority of clear-cell renal carcinomas, VHL function is lost, resulting in constitutive activation of HIF-1. After re-introduction of functional VHL, renal carcinoma cell lines are no longer tumorigenic, but can be made tumorigenic by expression of HIF2a in which the prolyl residues that are subject to hydroxylation have been mutated. In addition to VHL loss-of-function, many other genetic alterations that inactivate tumor suppressors
Evaluation of HIF-1 inhibitors as anticancer agents
Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by upregulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.
Although many anticancer regimens have been introduced to date, their survival benefits are negligible, which is the reason that a more innovative treatment is required. Basically, the identification of the specific molecular features of tumor promotion has allowed for rational drug discovery in cancer treatment, and drugs have been screened based upon the modulation of specific molecular targets in tumor cells. Target-based drugs should satisfy the following two conditions.
First, they must act by a described mechanism.
Second, they must reduce tumor growth in vivo, associated with this mechanism.
Many key factors have been found to be involved in the multiple steps of cell growth signal-transduction pathways. Targeting these factors offers a strategy for preventing tumor growth; for example, competitors or antibodies blocking ligand–receptor interaction, and receptor tyrosine kinase inhibitors, downstream pathway inhibitors (i.e., RAS farnesyl transferase inhibitors, mitogen-activated protein kinase and mTOR inhibitors), and cell-cycle arresters (i.e., cyclin-dependent kinase inhibitors) could all be used to inhibit tumor growth.
In addition to the intracellular events, tumor environmental factors should be considered to treat solid tumors. Of these, hypoxia is an important cancer-aggravating factor because it contributes to the progression of a more malignant phenotype, and to the acquisition of resistance to radiotherapy and chemotherapy. Thus, transcription factors that regulate these hypoxic events are good targets for anticancer therapy and in particular HIF-1 is one of most compelling targets. In this paper, we introduce the roles of HIF-1 in tumor promotion and provide a summary of new anticancer strategies designed to inhibit HIF-1 activity.
Classical work in tumor cell metabolism focused on bioenergetics, particularly enhanced glycolysis and suppressed oxidative phosphorylation (the ‘Warburg effect’). But the biosynthetic activities required to create daughter cells are equally important for tumor growth, and recent studies are now bringing these pathways into focus. In this review, we discuss how tumor cells achieve high rates of nucleotide and fatty acid synthesis, how oncogenes and tumor suppressors influence these activities, and how glutamine metabolism enables macromolecular synthesis in proliferating cells.
Otto Warburg’s demonstration that tumor cells rapidly use glucose and convert the majority of it to lactate is still the most fundamental and enduring observation in tumor metabolism. His work, which ushered in an era of study on tumor metabolism focused on the relationship between glycolysis and cellular bioenergetics, has been revisited and expanded by generations of tumor biologists. It is now accepted that a high rate of glucose metabolism, exploited clinically by 18FDGPET scanning, is a metabolic hallmark of rapidly dividing cells, correlates closely with transformation, and accounts for a significant percentage of ATP generated during cell proliferation. A ‘metabolic transformation’ is required for tumorigenesis. Research over the past few years has reinforced this idea, revealing the conservation of metabolic activities among diverse tumor types, and proving that oncogenic mutations can promote metabolic autonomy by driving nutrient uptake to levels that often exceed those required for cell growth and proliferation.
In order to engage in replicative division, a cell must duplicate its genome, proteins, and lipids and assemble the components into daughter cells; in short, it must become a factory for macromolecular biosynthesis. These activities require that cells take up extracellular nutrients like glucose and glutamine and allocate them into metabolic pathways that convert them into biosynthetic precursors (Figure 1). Tumor cells can achieve this phenotype through changes in the expression of enzymes that determine metabolic flux rates, including nutrient transporters and enzymes [8– 10]. Current studies in tumor metabolism are revealing novel mechanisms for metabolic control, establishing which enzyme isoforms facilitate the tumor metabolic phenotype, and suggesting new targets for cancer therapy.
The ongoing challenge in tumor cell metabolism is to understand how individual pathways fit together into the global metabolic phenotype of cell growth. Here we discuss two biosynthetic activities required by proliferating tumor cells: production of ribose-5 phosphate for nucleotide biosynthesis and production of fatty acids for lipid biosynthesis. Nucleotide and lipid biosynthesis share three important characteristics.
First, both use glucose as a carbon source.
Second, both consume TCA cycle intermediates, imposing the need for a mechanism to replenish the cycle.
Third, both require reductive power in the form of NADPH.
In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1, not shown). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.
The Tumor Microenvironment Selects for Altered Metabolism One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).
Blood vessels recruited to the tumor microenvironment, however, are disorganized, may not deliver blood effectively, and therefore do not completely alleviate hypoxia (reviewed in Gatenby and Gillies, 2004). The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment. Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF. Other molecular mechanisms are likely to be important, such as the metabolic changes induced by oncogene activation and tumor suppressor loss.
Oncogene Activation Drives Changes in Metabolism Not only may the tumor microenvironment select for a deranged metabolism, but oncogene status can also drive metabolic changes. Since Warburg’s time, the biochemical study of cancer metabolism has been overshadowed by efforts to identify the mutations that contribute to cancer initiation and progression. Recent work, however, has demonstrated that the key components of the Warburg effect—
increased glucose consumption,
decreased oxidative phosphorylation, and
accompanying lactate production—
are also distinguishing features of oncogene activation.
The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Ramanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes.
Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty acid synthesis and TCA cycle glucose oxidation. This primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment.
Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.
Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)
László G. Boros, Daniel J. Brackett and George G. Harrigan
Current Cancer Drug Targets, 2003, 3, 447-455 447
Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150 kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, while silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.
A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans
Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.
Review ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference
Sara Tomaselli, Barbara Bonamassa, Anna Alisi, Valerio Nobili, Franco Locatelli and Angela Gallo
Int. J. Mol. Sci. 19 Nov 2013; 14, 22796-22816 http://dx.doi.org:/10.3390/ijms141122796
The fermented wheat germ extract (FWGE) nutraceutical (Avemar™), manufactured under “good manufacturing practice” conditions and, fulfilling the self-affirmed “generally recognized as safe” status in the United States, has been approved as a “dietary food for special medical purposes for cancer patients” in Europe. In this paper, we report the adjuvant use of this nutraceutical in the treatment of high-risk skin melanoma patients. Methods: In a randomized, pilot, phase II clinical trial, the efficacy of dacarbazine (DTIC)-based adjuvant chemotherapy on survival parameters of melanoma patients was compared to that of the same treatment supplemented with a 1-year long administration of FWGE. Results: At the end of an additional 7-year-long follow-up period, log-rank analyses (Kaplan-Meier estimates) showed significant differences in both progression-free (PFS) and overall survival (OS) in favor of the FWGE group. Mean PFS: 55.8 months (FWGE group) versus 29.9 months (control group), p 0.0137. Mean OS: 66.2 months (FWGE group) versus 44.7 months (control group), p < 0.0298. Conclusions: The inclusion of Avemar into the adjuvant protocols of high-risk skin melanoma patients is highly recommended.
Adjuvant Fermented Wheat Germ Extract (Avemar™) Nutraceutical Improves Survival of High-Risk Skin Melanoma Patients: A Randomized, Pilot, Phase II Clinical Study with a 7-Year Follow-Up
LV Demidov, LV Manziuk, GY Kharkevitch, NA Pirogova, and EV Artamonova
Cancer Biotherapy & Radiopharmaceuticals 2008; 23(4) http://dx.doi.org:/10.1089/cbr.2008.0486
Cancer cells possess unique metabolic signatures compared to normal cells, including shifts in aerobic glycolysis, glutaminolysis, and de novo biosynthesis of macromolecules. Targeting these changes with agents (drugs and dietary components) has been employed as strategies to reduce the complications associated with tumorigenesis. This paper highlights the ability of several food components to suppress tumor-specific metabolic pathways, including increased expression of glucose transporters, oncogenic tyrosine kinase, tumor-specific M2-type pyruvate kinase, and fatty acid synthase, and the detection of such effects using various metabonomic technologies, including liquid chromatography/mass spectrometry (LC/MS) and stable isotope-labeled MS. Stable isotope-mediated tracing technologies offer exciting opportunities for defining specific target(s) for food components. Exposures, especially during the early transition phase from normal to cancer, are critical for the translation of knowledge about food components into effective prevention strategies. Although appropriate dietary exposures needed to alter cellular metabolism remain inconsistent and/or ill-defined, validated metabonomic biomarkers for dietary components hold promise for establishing effective strategies for cancer prevention.
Bioactive Food Components and Cancer-Specific Metabonomic Profiles
This reviewer poses the following observation. The importance of the pyridine nucleotide reduced/oxidized ratio has not been alluded to here, but the importance cannot be understated. It has relevance to the metabolic functions of anabolism and catabolism of the visceral organs. The importance of this has ties to the pentose monophosphate pathway. The importance of the pyridine nucleotide transhydrogenase reaction remains largely unexplored. In reference to the NAD-redox state, the observation was made by Nathan O. Kaplan that the organs may be viewed with respect to their primary functions in anabolic or high energy catabolic activities. Thus we find that the endocrine organs are largely tied to anabolic functioning, and to NADP, whereas cardiac and skeletal muscle are highly dependent on NAD. The consequence of this observed phenomenon appears to be related to a difference in the susceptibility to malignant transformation. In the case of the gastrointestinal tract, the rate of turnover of the epithelium is very high. However, with the exception of the liver, there is no major activity other than cell turnover. In the case of the liver, there is a major commitment to synthesis of lipids, storage of fuel, and synthesis of proteins, which is largely anabolic, but there is also a major activity in detoxification, which is not. In addition, the liver has a double circulation. As a result, a Zahn infarct is uncommon. Now we might also consider the heart. The heart is a muscle syncytium with a high need for oxygen. Cutting of the oxygen supply makes the myocytes vulnerable to ischemic insult and abberant rhythm abnormalities. In addition, the cardiomyocyte can take up lactic acid from the circulation for fuel, which is tied to the utilization of lactate from vigorous skeletal muscle activity. The skeletal muscle is tied to glycolysis in normal function, which has a poor generation of ATP, so that the recycling of excess lactic acid is required by cardiac muscle and hepatocytes. This has not been a part of the discussion, but this reviewer considers it important to remember in considering the organ-specific tendencies to malignant transformation.
Otto Warburg observed that many cancers lose their capacity for mitochondrial respiration, limiting ATP production to anaerobic glycolytic pathways. The phenomenon is particularly prevalent in aggressive malignancies, most of which are also hypoxic [1].
Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines [2-3]. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines [4].
Hypoxia reduces the ability of cells to maintain their energy levels, because less ATP is obtained from glycolysis than from oxidative phosphorylation. Cells adapt to hypoxia by activating the expression of mutant genes in glycolysis.
-Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.
The most direct induction of apoptosis caused by hypoxia is determined by the inhibition of the electron carrier chain from the inner membrane of the mitochondria. The lack of oxygen inhibits the transport of protons and thereby causes a decrease in membrane potential. Cell survival under conditions of mild hypoxia is mediated by phosphoinositide-3 kinase (PIK3) using severe hypoxia or anoxia, and then cells initiate a cascade of events that lead to apoptosis [5].
After DNA damage, a very important regulator of apoptosis is the p53 protein. This tumor suppressor gene has mutations in over 60% of human tumors and acts as a suppressor of cell division. The growth-suppressive effects of p53 are considered to be mediated through the transcriptional trans-activation activity of the protein. In addition to the maturational state of the clonal tumor, the prognosis of patients with CLL is dependent of genetic changes within the neoplastic cell population.
1.Warburg O. On the origin of cancer cells. Science 1956; 123 (3191):309-314
PubMed Abstract ; Publisher Full Text
2.Giardina TM, Steer JH, Lo SZ, Joyce DA. Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages. Biochim Biophys Acta 2008, 1777(2):118-129. PubMed Abstract | Publisher Full Text
3. Horimoto M, Resnick MB, Konkin TA, Routhier J, Wands JR, Baffy G. Expression of uncoupling protein-2 in human colon cancer. Clin Cancer Res 2004; 10 (18 Pt1):6203-6207. PubMed Abstract | Publisher Full Text
4. Randle PJ, England PJ, Denton RM. Control of the tricarboxylate cycle and it interactions with glycolysis during acetate utilization in rat heart. Biochem J 1970; 117(4):677-695. PubMed Abstract | PubMed Central Full Text
5. Gillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008; 49(Suppl 2):24S-42S. PubMed Abstract | Publisher Full Text
Shortened version of Comment –
Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines.
Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.
Hormonal Therapy, Complementary and Alternative Therapies – 9.4
Writer and Curator: Larry H. Bernstein, MD, FCAP
The following material has been covered from a different vantage point in previous writings. In the previous articles the focus was both antibacterial and anticancer resistance. Much of the focus was on the metabolomic studies of substrate fluxes across cells and extracellular analyses to determine differences between cancer cell types in vitro. This time I shall cover some of the selected ground with a different perspective. Alternative medicines have been around for thousands of years. Much of it was based on readily available plants that had a pharmacological action. This also gave rise to pharmaceuticals by extraction of the active compound. Such was the case with digitalis and also with Warfarin.
9.4.1 Use of complementary medicine by adult patients participating in cancer clinical trials
9.4.2 Complementary/Alternative Medicine Use in a Comprehensive Cancer Center and the Implications for Oncology
9.4.3 Trends in Alternative Medicine Use in the United States, 1990-1997 Results of a Follow-up National Survey
9.4.4 Courses Involving Complementary and Alternative Medicine at US Medical Schools
PURPOSE/OBJECTIVES: To document the prevalence, demographic correlates, patterns of use, and beliefs about complementary and alternative medicine (CAM) therapies of adult patients enrolled in National Cancer Institute (NCI) clinical trials. DESIGN: Prospective, cross-sectional, descriptive survey.
SETTING: W.G. Magnuson Clinical Center of the National Institutes of Health in Bethesda, MD. SAMPLE: Convenience sample of 100 English-speaking, adult patients with cancer admitted to intramural clinical trials.
METHODS: A standardized, 99-item questionnaire assessing use of CAM therapies pre- and postcancer diagnosis was administered by face-to-face interview. MAIN RESEARCH VARIABLES: Use of CAM therapies, beliefs, communication with physician. FINDINGS: 63% used at least one CAM therapy, with an average use of two therapies per patient. Men were significantly less likely to use a therapy than women; women were more likely to use numerous therapies. Cancer diagnosis seems to have had no influence overall on the frequency of use of CAM therapies. The major reasons stated for CAM use were for treatment-related medical conditions as well as depression, anxiety, and insomnia. The most frequently reported therapies were spiritual, relaxation, imagery, exercise, lifestyle diet (e.g., macrobiotic, vegetarian), and nutritional supplementation. Patients unanimously believed that these complementary therapies helped to improve their quality of life through more effective coping with stress, decreasing the discomforts of treatment and illness, and giving them a sense of control. CONCLUSIONS: Patients with cancer use various complementary therapies to cope with their disease and the rigors of clinical trials. Women and those with higher educational backgrounds were more frequent users.
IMPLICATIONS FOR NURSING PRACTICE: Nurses who provide care to subjects of biomedical research have an opportunity and responsibility regarding their patients’ use of CAM therapies. Nurses may use in-house resources to help evaluate subjects’ use of a CAM modality or to provide quality-of-life therapies such as relaxation, imagery, or healing touch. Discussing these health practices in a nonjudgmental manner adds to the assessment of patients’ coping skills and ability to make decisions about their health care.
Purpose: Oncologists are aware that their patients use complementary/ alternative medicine (CAM). As cancer incidence rates and survival time increase, use of CAM will likely increase. This study assessed the prevalence and predictors of CAM use in a comprehensive cancer center. Subjects and Methods: Subjects were English-speaking cancer patients at least 18 years of age, attending one of eight outpatient clinics at The University of Texas M.D. Anderson Cancer Center, Houston, TX, between December 1997 and June 1998. After giving written informed consent, participants completed a self-administered questionnaire. Differences between CAM users and nonusers were assessed by Chi square and univariate logistic regression analysis. A multivariate logistic regression model identified the simultaneous impact of demographic, clinical, and treatment variables on CAM use; P values were two-sided. Results: Of the 453 participants (response rate, 51.4%), 99.3% had heard of CAM. Of those, 83.3% had used at least one CAM approach. Use was greatest for spiritual practices (80.5%), vitamins and herbs (62.6%), and movement and physical therapies (59.2%) and predicted (P < .001) by sex (female), younger age, indigent pay status, and surgery. After excluding spiritual practices and psychotherapy, 95.8% of participants were aware of CAM and 68.7% of those had used CAM. Use was predicted (P < .0001) by sex (female), education, and chemotherapy. Conclusion: In most categories, CAM use was common among outpatients. Given the number of patients combining vitamins and herbs with conventional treatments, the oncology community must improve patient provider communication, offer reliable information to patients, and initiate research to determine possible drug-herb-vitamin interactions.
9.4.3Trends in Alternative Medicine Use in the United States, 1990-1997 Results of a Follow-up National Survey
DM Eisenberg, RB Davis, SL Ettner, S Appel, S Wilkey, M Van Rompay; RC Kessler JAMA. 1998; 280(18):1569-1575. http://dx.doi.org:/10.1001/jama.280.18.1569
Context. A prior national survey documented the high prevalence and costs of alternative medicine use in the United States in 1990. Objective. To document trends in alternative medicine use in the United States between 1990 and 1997. Design. Nationally representative random household telephone surveys using comparable key questions were conducted in 1991 and 1997 measuring utilization in 1990 and 1997, respectively. Participants. A total of 1539 adults in 1991 and 2055 in 1997. Main Outcomes Measures. Prevalence, estimated costs, and disclosure of alternative therapies to physicians. Results. Use of at least 1 of 16 alternative therapies during the previous year increased from 33.8% in 1990 to 42.1% in 1997 (P≤.001). The therapies increasing the most included: herbal medicine, massage, megavitamins, self-help groups, folk remedies, energy healing, and homeopathy. The probability of users visiting an alternative medicine practitioner increased from 36.3% to 46.3% (P=.002). In both surveys alternative therapies were used most frequently for chronic conditions, including back problems, anxiety, depression, and headaches. There was no significant change in disclosure rates between the 2 survey years; 39.8% of alternative therapies were disclosed to physicians in 1990 vs 38.5% in 1997. The percentage of users paying entirely out-of-pocket for services provided by alternative medicine practitioners did not change significantly between 1990 (64.0%) and 1997 (58.3%) (P=.36). Extrapolations to the US population suggest a 47.3% increase in total visits to alternative medicine practitioners, from 427 million in 1990 to 629 million in 1997, thereby exceeding total visits to all US primary care physicians. An estimated 15 million adults in 1997 took prescription medications concurrently with herbal remedies and/or high-dose vitamins (18.4% of all prescription users). Estimated expenditures for alternative medicine professional services increased 45.2% between 1990 and 1997 and were conservatively estimated at $21.2 billion in 1997, with at least $12.2 billion paid out-of-pocket. This exceeds the 1997 out-of-pocket expenditures for all US hospitalizations. Total 1997 out-of-pocket expenditures relating to alternative therapies were conservatively estimated at $27.0 billion, which is comparable with the projected 1997 out-of-pocket expenditures for all US physician services. Conclusions. Alternative medicine use and expenditures increased substantially between 1990 and 1997, attributable primarily to an increase in the proportion of the population seeking alternative therapies, rather than increased visits per patient.
9.4.4Courses Involving Complementary and Alternative Medicine at US Medical Schools Miriam S. Wetzel, David M. Eisenberg, Ted J. Kaptchuk JAMA. 1998; 280(9):784-787. http://dx.doi.org:/10.1001/jama.280.9.784
Context. With the public’s increasing use of complementary and alternative medicine, medical schools must consider the challenge of educating physicians about these therapies. Objectives. To document the prevalence, scope, and diversity of medical school education in complementary and alternative therapy topics and to obtain information about the organizational and academic features of these courses. Design. Mail survey and follow-up letter and telephone survey conducted in 1997-1998. Participants. Academic or curriculum deans and faculty at each of the 125 US medical schools. Main Outcome Measures. Courses taught at US medical schools and administrative and educational characteristics of these courses. Results. Replies were received from 117 (94%) of the 125 US medical schools. Of schools that replied, 75 (64%) reported offering elective courses in complementary or alternative medicine or including these topics in required courses. Of the 123 courses reported, 84 (68%) were stand-alone electives, 38 (31%) were part of required courses, and one (1%) was part of an elective. Thirty-eight courses (31%) were offered by departments of family practice and 14 (11%) by departments of medicine or internal medicine. Educational formats included lectures, practitioner lecture and/or demonstration, and patient presentations. Common topics included chiropractic, acupuncture, homeopathy, herbal therapies, and mind-body techniques. Conclusions. There is tremendous heterogeneity and diversity in content, format, and requirements among courses in complementary and alternative medicine at US medical schools.
More than one-third of adults and nearly 12 percent of children in the United States use alternatives to traditional medicine, according to a large federal survey released today that documents how entrenched acupuncture, herbal remedies and other once-exotic therapies have become.
The 2007 survey of more than 32,000 Americans, which for the first time included children, found that use of yoga, “probiotics,” fish oil and other “complementary and alternative” therapies held steady among adults since the last national survey five years earlier, and that such treatments have become part of health care for many youngsters.
“It’s clear that millions of Americans every year are turning to complementary and alternative medicine,” said Richard L. Nahin of the National Institutes of Health’s National Center for Complementary and Alternative Medicine, which released the survey. “The use of complementary and alternative medicine seems to have stabilized in the United States.”
The most commonly used are dietary supplements and herbal products such as echinacea, flaxseed oil and ginseng, followed by deep-breathing exercises, meditation, chiropractic therapy, massage and yoga. Although fewer Americans were using certain diets and trying herbal remedies such as echinacea to cure colds, the popularity of acupuncture, meditation, yoga and massage grew. 9.4.6Wheat germ extract (Avemar, Avé, AvéULTRA, AWGE, OncoMAR) http://mskcc.org/cancer-care/herb/wheat-germ-extract
Fermented wheat germ extract (WGE) was developed in the 1990s by Hungarian chemist Mate Hidvegi. It should not be confused with wheat germ oil. WGE is used as a dietary supplement by cancer patients in Hungary to improve quality of life (QoL).
Results from in vitro studies show that WGE has anticancer (1)(2)(3)(4)(5)(19), antimetastatic (6), and immunomodulatory (2)(7) effects. Although it appears to increase estrogen receptor (ER) activity, WGE enhanced efficacy of tamoxifen, an ER antagonist, in ER+ breast cancer cells (8) as well as cisplatin in ovarian cancer cell lines (5). Animal models suggest WGE can reduce cardiovascular symptoms due to chronic hypertension, diabetes, and obesity (9), mitigate symptoms associated with lupus (10), and that its antitumor effect is comparable to other endocrine treatments (11).
Data from pilot studies indicate a beneficial role for WGE in patients with colorectal cancer (12) and in reducing treatment-associated febrile neutropenia in pediatric cancer patients (13). It also prolonged survival of patients with melanoma when used with chemotherapy (14)(15). However, these effects must be confirmed by large-scale, well-designed clinical trials.
Because it potentiates estrogen receptor activity, patients with hormone-sensitive cancers should use WGE with caution.
Avemar is an all-natural, clinically proven, dietary supplement for cancer patients. Medical experts recognize Avemar as an effective supportive cancer treatment and recommend complementing the diet of concerned patients with Avemar.Scientific studies showed that Avemar also enhances the efficacy of conventional oncological treatments (surgery, radio-, chemo- and immunotherapy). With regular intake of Avemar, patients can relish a better quality of life and can enjoy a better lifestyle. Avemar is available in granulate and film-coated tablet form.
The active ingredient is Avemar pulvis, an all-natural compound made from fermented wheat germ extract using patented biotechnological processes. Since the invention of Avemar pulvis significant research has been undertaken – not only in the laboratory, but in test animals and human cancer patients as well. Numerous scientific studies have been conducted to study its safety profile and its effectiveness, including in vitro, in vivo and human clinical research. Over 100 reports have been written for presentation or publication since 1998, and over 33 peer-reviewed scientific papers are currently accessible at PubMed database. All scientific publications are accessible at the official Avemar Research website.
Avemar is a naturally fermented wheat germ extract that has been subjected to a great deal of research scrutiny, particularly in the area of cancer treatment. What makes Avemar stand out among other known therapies is the fact that its effectiveness isn’t limited to any one specific type of cancer. So far it has exhibited positive effects against all forms of cancer cell lines tested.
Whether cancer cells proceed to replicate, grow, and eventually spread throughout the body is determined by enzymatic activity and their accessibility to various nutrients.
Pharmaceutical companies have focused their efforts to find cures for various forms of cancer. One of their top priorities (and one area with the greatest potential) has always been to uncover compounds that inhibit glucose metabolism in tumor cells.
Every form of cancer cell utilizes glucose at rates 10 to 50 times higher than that of normal healthy cells (a well-known phenomenon referred to as “the Warburg effect”). Unlike normal, healthy cells that utilize glucose primarily for energy, tumor cells use glucose to replicate cells. They convert glucose to nucleic acids (necessary for the formation of additional RNA) by a hexose monophosphate pathway. They also have to break down tissue in order to make proteins (needed for the cancer to continue to grow). This is termed cachexia. In this sense the behavior of cancer proliferation is like a systemic infection.
In simple terms, cancer cells have only one function: proliferation. To achieve this function, cancer cells need large amounts of glucose that they can convert into building materials for new cells. As the tumor grows, more and more glucose is consumed.
Research indicates that Avemar works through several different mechanisms. One of its most unique benefits, however, is its ability to inhibit glucose metabolism in cancer cells.
Research at UCLA has demonstrated that Avemar reduces glucose flow into cancer cells—which inhibits their ability to produce additional nucleic acids and subsequently reduces their proliferation or growth. In the presence of Avemar compounds, cancer cells begin to utilize the available glucose to produce substances that actually inhibit cell division and stimulate programmed cell death (apoptosis) within the tumor.
As one report explains, decreased glucose consumption of the tumors results in a harmonizing of the patient’s metabolism—as well as weight gain, even in people with advanced cancers. As a result, patients treated with Avemar also have improved tolerance for surgery, radiation, and chemotherapy. Further, Avemar achieves these results without creating any toxicity or damage to normal, healthy cells. (Ann N Y Acad Sci. 07;1110:348–61)
This particular feature of Avemar explains why cancer patients using the product routinely experience an improved quality of life. They have less fatigue, pain, and depression, and experience an increase in appetite that can help them regain lost weight. (Medicus Anonymus/Pulmono 03;11 (Suppl 1):13–14) (24th Congress of the Hungarian Cancer Society, Budapest, Hungary 2001)
* Importantly, the spice can stop the action of the enzyme COX-2 known to produce negative, inflammation causing localised enzymes (eicosanoids). Such inflammation is a known precursor to cancer.
* It has also been shown to inhibit vascular epithelial growth factors. Every tumor needs a blood supply – the growth factors build one, but curcumin seems to stop them.
* It has been shown to ´re-awaken´ a key tumor suppressor gene.
* It has been shown to inhibit metastases.
* It has been shown to kill cancer cells (B lymphoma cells).
* It prevents regrowth of cancer stem cells which lie at the heart of many tumors
In the journal ´Genes and Nutrition´ (2011; 6(2):93-108) the whole issue of ´Epigenetics´ was exposed. It used to be thought that your genes controlled all and a problem in a gene meant you were in some way ´doomed´. This theory has been disproven and replaced by one that shows genes are just your blueprint; these blueprints are controlled, activated or suppressed by the localized environment. So hormones can affect their action, as can natural compounds in food. And curcumin seems to affect gene expression significantly. Such ´signaling pathways´ have been shown to be affected by curcumin.
An example of this ´signaling pathway´ modification came in research from the Ludwig-Maximilians University in Munich, Germany in 2012 which showed that curcumin can inhibit the formation of metastases in both prostate and breast cancer. Both cancers spread throughout the body through the release of chemical messengers, pro-inflammatory cytokines CXCL1 and CXCL2, but curcumin alters the expression of these two damaging proteins.
Next, Cheryl Myers (head of Scientific Affairs and Education for EuroPharma Inc.) refers to curcumin as ´the anti-cancer herb´ because of its success in stopping cancer formation, replication and spread. Research also shows that curcumin increases the activity of certain anti-cancer drugs while protecting healthy cells and organs. It has been proven to reduce systemic inflammation and oxidative stress.
And researchers from the Dept. of Natural Science at Middlesex University have shown that curcumin and chokeberry can work together to induce cancer cell death (apoptosis) and stop the spread of malignant cancer cells. Their report (in Oncology Reports) was for brain tumors.
Dr Young S. Kim leading a team at the National Cancer Institute in America showed that curcumin was one of the natural compounds that could prevent cancer stem cells from re-growing and re-forming the cancer. Her conclusion even suggested patients could supplement!
The University of Missouri has shown curcumin can counter the dangerous effects of HRT and its link to breast cancer cause. “The results of the study show that women could potentially take curcumin to protect themselves from developing progestin-accelerated tumors,” said the lead researcher. Synthetic progestin increases VEGF a protein that helps form blood supplies to developing tumors. Curcumin inhibits VEGF and thus reduces the potential of breast cancer to grow.
Professor Bharat Aggarwal Ph. D. in MD Anderson Department of Therapeutics has conducted a number of studies, for example showing that in a Phase II clinical trial, pancreatic cancer patients having no chemotherapy, it reduced tumor size. He believes it is effective against many types of cancer because it suppresses angiogenesis (the growth of blood vessels essential to a tumor). “The reason curcumin is so effective against cancer is that it hits not just a single target or cell signaling pathway but dozens of targets implicated in cancer.” It has also been shown to have a strong synergistic effect against cancer with resveratrol, and also with EGCG in green tea.
What is exciting the experts in cancer centers in America is that it can play a role against several of the steps in what is a multi-step cancer process. As such it would seem stupid to ignore it as a part of an Integrative or holistic cancer treatment program.
What are curcumin and turmeric?
Curcumin is the active ingredient of the Indian/Asian curry spice Turmeric. To put this technically, curcumin is the principal curcuminoid in turmeric. Curcuminoids are polyphenols. Turmeric powder is ground from the root of a plant called Curcuma Longa, which is a member of the ginger family and is found throughout Southern Asia, even growing wild in the Himalayas.
This vivid yellow to brown spice was used, like many Asian spices and chillies, to hide the taste of stronger tasting meats and fish even those that might have gone a little off in such hot climates. Like many such spices, it also performed a necessary and functional role it was a cleanser, a bacteria-killer in the stomach, protecting against tainted foods!
Curcumin/turmeric has been used in Ayurvedic medicine for thousands of years as a cleanser of the body. It appears to work at a number of levels:
It can inhibit unwanted bacterial action in the stomach and intestine:
For example (i) , University of Chicago researchers have shown it inhibits Helicobacter pylori, a bacterium known to be responsible for stomach ulcers and some stomach cancers. In Ayurvedic medicine, curcumin was used in poultices for this same reason to kill unwanted bacteria.
It is a significant anti-inflammatory:
Arachidonic acid is a precursor/stimulator of the production of bad eicosancids (see our reviews of omega 3 and vitamin D) and thus to inflammation, which is itself a precursor to certain cancers. Curcumin has been found to inhibit several of the pre-inflammatory enzymes (e.g. COX2 and iNOS) in vitro and in vivo with animals. Japanese research suggests it works in much the same way as salicylin.
It boosts crucial cellular glutathione levels:
Glutathione is a crucial intracellular antioxidant, helping the cell maintain its correct oxygen levels and fight off the effects of stress hormones. Research has shown that curcumin can prevent the action of an enzyme that limits glutathione production.
It is a powerful antioxidant:
Turmeric extract tested more potent than garlic, omega 3 and cat´s claw (devil´s claw) said German research.
It can help prevent liver damage
2010 research from St Louis has shown that it can turn off a protein called Leptin, which causes liver damage. It has also been shown to be capable of detoxifying the liver. Thus curcumin may be of help in keeping the liver healthy during chemotherapy cancer treatments.
It can prevent and even ´treat´ cancer:
As we have covered above, curcumin can suppress tumor initiation, promotion and metastasis. Extensive research over the last 50 years has indicated it can prevent and treat cancer. The anti-cancer potential stems from its ability to control gene signaling, and affect a wide variety of tumor cells, down-regulate transcription factors, down-regulate enzymes such as COX-2 and other inflammatories, cytokines, chemokines, cell-surface adhesion molecules, down-regulate growth factors, etc., etc.
Tufts have conducted research with breast cancer patients concluding that curcumin and isoflavanoids seem to inhibit the action of environmental estrogens.
UCLA have researched its potential with colorectal cancer (San Diego, Chauhen). And there are Clinical Trials underway (according to the Mayo Clinic to investigate curcumin as a way to prevent cancer in people with precancerous conditions, as a cancer treatment, and as a remedy for signs and symptoms caused by cancer treatments.
Much of the work original used cell cultures. Increasingly studies use a variety of animals, and there have been human trials, even clinical trials, primarily with cervical cancer lesions and with gastrointestinal cancers. So, although the biochemical knowledge is vast, the use of oral curcumin to prevent and treat cancer is still in its infancy.
Next, there is a problem maintaining effectiveness inside the cells; there are several studies that show oral consumption needs to be maintained in order to maintain blood and cellular levels. But it is not as simple as curry every day!
Curcumin supplements may be heavily contaminated with everything from pesticides to other spices so you must choose a reliable supplier, ideally of curcuma longa. If you are thinking of buying Curcuma Longa you might like to look at the Natural Selection as they are based in the UK and they tend to have natural compounds that avoid pesticides. Click here. Alternatively in the USA you might like to go to the Natural News or Dr Mercola websites.
Counter indications
Some supplements contain piperine, supposedly to increase bio-availability. This can interfere with certain drugs.
Curcumin can inhibit coagulation in vitro and so may be inadvisable if you are taking anti-coagulants. It might increase the risk of bleeding.
It might also be advisable not to take it if you are pregnant or lactating.
In summary
Clearly there is a huge enthusiasm, even expectation, overseas for curcumin, if not in British hospitals and oncology departments. But the real issue is can you take enough of it orally to deliver it in adequate doses to your breast or prostate cells? Certainly curcumin (curcuma longa) was seen to be an important herb in the natural and successful treatment of prostate cancer that we covered in Cancer Watch (icon 2006, issue 3 ). We will just have to wait and see whether this longstanding Ayurvedic medicine will curry favour with the UK medical fraternity.
Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer
Author and Curator: Larry H. Bernstein, MD, FCAP
This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to pharmaceutical discovery and personalized medicine. We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed. I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.
Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.
While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.
The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.
As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.
Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.
As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.
Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor. ….
As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.
Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.
Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits. …
The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.
The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.
A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion. THE SCIENTIST STAFF
From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.
Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.
This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.
Gut Microbiome Heritability
Analyzing data from a large twin study, researchers have homed in on how host genetics can shape the gut microbiome.
By Tracy Vence | The Scientist Nov 6, 2014
Previous research suggested host genetic variation can influence microbial phenotype, but an analysis of data from a large twin study published in Cell today (November 6) solidifies the connection between human genotype and the composition of the gut microbiome. Studying more than 1,000 fecal samples from 416 monozygotic and dizygotic twin pairs, Cornell University’s Ruth Ley and her colleagues have homed in on one bacterial taxon, the family Christensenellaceae, as the most highly heritable group of microbes in the human gut. The researchers also found that Christensenellaceae—which was first described just two years ago—is central to a network of co-occurring heritable microbes that is associated with lean body mass index (BMI). …
Of particular interest was the family Christensenellaceae, which was the most heritable taxon among those identified in the team’s analysis of fecal samples obtained from the TwinsUK study population.
While microbiologists had previously detected 16S rRNA sequences belonging to Christensenellaceae in the human microbiome, the family wasn’t named until 2012. “People hadn’t looked into it, partly because it didn’t have a name . . . it sort of flew under the radar,” said Ley.
Ley and her colleagues discovered that Christensenellaceae appears to be the hub in a network of co-occurring heritable taxa, which—among TwinsUK participants—was associated with low BMI. The researchers also found that Christensenellaceae had been found at greater abundance in low-BMI twins in older studies.
To interrogate the effects of Christensenellaceae on host metabolic phenotype, the Ley’s team introduced lean and obese human fecal samples into germ-free mice. They found animals that received lean fecal samples containing more Christensenellaceae showed reduced weight gain compared with their counterparts. And treatment of mice that had obesity-associated microbiomes with one member of the Christensenellaceae family, Christensenella minuta, led to reduced weight gain. …
Ley and her colleagues are now focusing on the host alleles underlying the heritability of the gut microbiome. “We’re running a genome-wide association analysis to try to find genes—particular variants of genes—that might associate with higher levels of these highly heritable microbiota. . . . Hopefully that will point us to possible reasons they’re heritable,” she said. “The genes will guide us toward understanding how these relationships are maintained between host genotype and microbiome composition.”
The desire for temporal and spatial control of medications to minimize side effects and maximize benefits has inspired the development of light-controllable drugs, or optopharmacology. Early versions of such drugs have manipulated ion channels or protein-protein interactions, “but never, to my knowledge, G protein–coupled receptors [GPCRs], which are one of the most important pharmacological targets,” says Pau Gorostiza of the Institute for Bioengineering of Catalonia, in Barcelona.
Gorostiza has taken the first step toward filling that gap, creating a photosensitive inhibitor of the metabotropic glutamate 5 (mGlu5) receptor—a GPCR expressed in neurons and implicated in a number of neurological and psychiatric disorders. The new mGlu5 inhibitor—called alloswitch-1—is based on a known mGlu receptor inhibitor, but the simple addition of a light-responsive appendage, as had been done for other photosensitive drugs, wasn’t an option. The binding site on mGlu5 is “extremely tight,” explains Gorostiza, and would not accommodate a differently shaped molecule. Instead, alloswitch-1 has an intrinsic light-responsive element.
In a human cell line, the drug was active under dim light conditions, switched off by exposure to violet light, and switched back on by green light. When Gorostiza’s team administered alloswitch-1 to tadpoles, switching between violet and green light made the animals stop and start swimming, respectively.
The fact that alloswitch-1 is constitutively active and switched off by light is not ideal, says Gorostiza. “If you are thinking of therapy, then in principle you would prefer the opposite,” an “on” switch. Indeed, tweaks are required before alloswitch-1 could be a useful drug or research tool, says Stefan Herlitze, who studies ion channels at Ruhr-Universität Bochum in Germany. But, he adds, “as a proof of principle it is great.” (Nat Chem Biol, http://dx.doi.org:/10.1038/nchembio.1612, 2014)
To understand disease processes, scientists often focus on unraveling how gene expression in disease-associated cells is altered. Increases or decreases in transcription—as dictated by a regulatory stretch of DNA called an enhancer, which serves as a binding site for transcription factors and associated proteins—can produce an aberrant composition of proteins, metabolites, and signaling molecules that drives pathologic states. Identifying the root causes of these changes may lead to new therapeutic approaches for many different diseases.
Although few therapies for human diseases aim to alter gene expression, the outstanding examples—including antiestrogens for hormone-positive breast cancer, antiandrogens for prostate cancer, and PPAR-γ agonists for type 2 diabetes—demonstrate the benefits that can be achieved through targeting gene-control mechanisms. Now, thanks to recent papers from laboratories at MIT, Harvard, and the National Institutes of Health, researchers have a new, much bigger transcriptional target: large DNA regions known as super-enhancers or stretch-enhancers. Already, work on super-enhancers is providing insights into how gene-expression programs are established and maintained, and how they may go awry in disease. Such research promises to open new avenues for discovering medicines for diseases where novel approaches are sorely needed.
Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions (Cell, 153:307-19, 2013). They also appear to bind a large percentage of the transcriptional machinery compared to typical enhancers, allowing them to better establish and enforce cell-type specific transcriptional programs (Cell, 153:320-34, 2013).
Super-enhancers are closely associated with genes that dictate cell identity, including those for cell-type–specific master regulatory transcription factors. This observation led to the intriguing hypothesis that cells with a pathologic identity, such as cancer cells, have an altered gene expression program driven by the loss, gain, or altered function of super-enhancers.
Sure enough, by mapping the genome-wide location of super-enhancers in several cancer cell lines and from patients’ tumor cells, we and others have demonstrated that genes located near super-enhancers are involved in processes that underlie tumorigenesis, such as cell proliferation, signaling, and apoptosis.
Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions.
Genome-wide association studies (GWAS) have found that disease- and trait-associated genetic variants often occur in greater numbers in super-enhancers (compared to typical enhancers) in cell types involved in the disease or trait of interest (Cell, 155:934-47, 2013). For example, an enrichment of fasting glucose–associated single nucleotide polymorphisms (SNPs) was found in the stretch-enhancers of pancreatic islet cells (PNAS, 110:17921-26, 2013). Given that some 90 percent of reported disease-associated SNPs are located in noncoding regions, super-enhancer maps may be extremely valuable in assigning functional significance to GWAS variants and identifying target pathways.
Because only 1 to 2 percent of active genes are physically linked to a super-enhancer, mapping the locations of super-enhancers can be used to pinpoint the small number of genes that may drive the biology of that cell. Differential super-enhancer maps that compare normal cells to diseased cells can be used to unravel the gene-control circuitry and identify new molecular targets, in much the same way that somatic mutations in tumor cells can point to oncogenic drivers in cancer. This approach is especially attractive in diseases for which an incomplete understanding of the pathogenic mechanisms has been a barrier to discovering effective new therapies.
Another therapeutic approach could be to disrupt the formation or function of super-enhancers by interfering with their associated protein components. This strategy could make it possible to downregulate multiple disease-associated genes through a single molecular intervention. A group of Boston-area researchers recently published support for this concept when they described inhibited expression of cancer-specific genes, leading to a decrease in cancer cell growth, by using a small molecule inhibitor to knock down a super-enhancer component called BRD4 (Cancer Cell, 24:777-90, 2013). More recently, another group showed that expression of the RUNX1 transcription factor, involved in a form of T-cell leukemia, can be diminished by treating cells with an inhibitor of a transcriptional kinase that is present at the RUNX1 super-enhancer (Nature, 511:616-20, 2014).
detect pathogen-associated molecular patterns to activate immunity,
pathogens attempt to deregulate host immunity through secreted effectors.
Fungi employ LysM effectors to prevent
recognition of cell wall-derived chitin by host immune receptors
Structural analysis of the LysM effector Ecp6 of
the fungal tomato pathogen Cladosporium fulvum reveals
a novel mechanism for chitin binding,
mediated by intrachain LysM dimerization,
leading to a chitin-binding groove that is deeply buried in the effector protein.
This composite binding site involves
two of the three LysMs of Ecp6 and
mediates chitin binding with ultra-high (pM) affinity.
The remaining singular LysM domain of Ecp6 binds chitin with
low micromolar affinity but can nevertheless still perturb chitin-triggered immunity.
Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.
Mutated Genes in Schizophrenia Map to Brain Networks From www.nih.gov – Sep 3, 2013
Previous studies have shown that many people with schizophrenia have de novo, or new, genetic mutations. These misspellings in a gene’s DNA sequence
occur spontaneously and so aren’t shared by their close relatives.
Dr. Mary-Claire King of the University of Washington in Seattle and colleagues set out to
identify spontaneous genetic mutations in people with schizophrenia and
to assess where and when in the brain these misspelled genes are turned on, or expressed.
The study was funded in part by NIH’s National Institute of Mental Health (NIMH). The results were published in the August 1, 2013, issue of Cell.
The researchers sequenced the exomes (protein-coding DNA regions) of 399 people—105 with schizophrenia plus their unaffected parents and siblings. Gene variations
that were found in a person with schizophrenia but not in either parent were considered spontaneous.
The likelihood of having a spontaneous mutation was associated with
the age of the father in both affected and unaffected siblings.
Significantly more mutations were found in people
whose fathers were 33-45 years at the time of conception compared to 19-28 years.
Among people with schizophrenia, the scientists identified
54 genes with spontaneous mutations
predicted to cause damage to the function of the protein they encode.
The researchers used newly available database resources that show
where in the brain and when during development genes are expressed.
The genes form an interconnected expression network with many more connections than
that of the genes with spontaneous damaging mutations in unaffected siblings.
The spontaneously mutated genes in people with schizophrenia
were expressed in the prefrontal cortex, a region in the front of the brain.
The genes are known to be involved in important pathways in brain development. Fifty of these genes were active
mainly during the period of fetal development.
“Processes critical for the brain’s development can be revealed by the mutations that disrupt them,” King says. “Mutations can lead to loss of integrity of a whole pathway,
not just of a single gene.”
These findings support the concept that schizophrenia may result, in part, from
disruptions in development in the prefrontal cortex during fetal development.
James E. Darnell’s “Reflections”
A brief history of the discovery of RNA and its role in transcription — peppered with career advice
By Joseph P. Tiano
James Darnell begins his Journal of Biological Chemistry “Reflections” article by saying, “graduate students these days
have to swim in a sea virtually turgid with the daily avalanche of new information and
may be momentarily too overwhelmed to listen to the aging.
I firmly believe how we learned what we know can provide useful guidance for how and what a newcomer will learn.” Considering his remarkable discoveries in
RNA processing and eukaryotic transcriptional regulation
spanning 60 years of research, Darnell’s advice should be cherished. In his second year at medical school at Washington University School of Medicine in St. Louis, while
studying streptococcal disease in Robert J. Glaser’s laboratory, Darnell realized he “loved doing the experiments” and had his first “career advancement event.”
He and technician Barbara Pesch discovered that in vivo penicillin treatment killed streptococci only in the exponential growth phase and not in the stationary phase. These
results were published in the Journal of Clinical Investigation and earned Darnell an interview with Harry Eagle at the National Institutes of Health.
Darnell arrived at the NIH in 1956, shortly after Eagle shifted his research interest to developing his minimal essential cell culture medium, still used. Eagle, then studying cell metabolism, suggested that Darnell take up a side project on poliovirus replication in mammalian cells in collaboration with Robert I. DeMars. DeMars’ Ph.D.
adviser was also James Watson’s mentor, so Darnell met Watson, who invited him to give a talk at Harvard University, which led to an assistant professor position
at the MIT under Salvador Luria. A take-home message is to embrace side projects, because you never know where they may lead: this project helped to shape
his career.
Darnell arrived in Boston in 1961. Following the discovery of DNA’s structure in 1953, the world of molecular biology was turning to RNA in an effort to understand how
proteins are made. Darnell’s background in virology (it was discovered in 1960 that viruses used RNA to replicate) was ideal for the aim of his first independent lab:
exploring mRNA in animal cells grown in culture. While at MIT, he developed a new technique for purifying RNA along with making other observations
suggesting that nonribosomal cytoplasmic RNA may be involved in protein synthesis.
When Darnell moved to Albert Einstein College of Medicine for full professorship in 1964, it was hypothesized that heterogenous nuclear RNA was a precursor to mRNA.
At Einstein, Darnell discovered RNA processing of pre-tRNAs and demonstrated for the first time
that a specific nuclear RNA could represent a possible specific mRNA precursor.
In 1967 Darnell took a position at Columbia University, and it was there that he discovered (simultaneously with two other labs) that
mRNA contained a polyadenosine tail.
The three groups all published their results together in the Proceedings of the National Academy of Sciences in 1971. Shortly afterward, Darnell made his final career move
four short miles down the street to Rockefeller University in 1974.
Over the next 35-plus years at Rockefeller, Darnell never strayed from his original research question: How do mammalian cells make and control the making of different
mRNAs? His work was instrumental in the collaborative discovery of
splicing in the late 1970s and
in identifying and cloning many transcriptional activators.
Perhaps his greatest contribution during this time, with the help of Ernest Knight, was
the discovery and cloning of the signal transducers and activators of transcription (STAT) proteins.
And with George Stark, Andy Wilks and John Krowlewski, he described
cytokine signaling via the JAK-STAT pathway.
Darnell closes his “Reflections” with perhaps his best advice: Do not get too wrapped up in your own work, because “we are all needed and we are all in this together.”
GxGD proteases are a family of intramembranous enzymes capable of hydrolyzing
the transmembrane domain of some integral membrane proteins.
The GxGD family is one of the three families of
intramembrane-cleaving proteases discovered so far (along with the rhomboid and site-2 protease) and
includes the γ-secretase and the signal peptide peptidase.
Although only recently discovered, a number of functions in human pathology and in numerous other biological processes
have been attributed to γ-secretase and SPP.
Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo highlighted the latest findings on the structure and function of γ-secretase and SPP
in a recent minireview in The Journal of Biological Chemistry.
γ-secretase is involved in cleaving the amyloid-β precursor protein, thus producing amyloid-β peptide,
the main component of senile plaques in Alzheimer’s disease patients’ brains. The complete structure of mammalian γ-secretase is not yet known; however,
Tomita and Iwatsubo note that biochemical analyses have revealed it to be a multisubunit protein complex.
Its catalytic subunit is presenilin, an aspartyl protease.
In vitro and in vivo functional and chemical biology analyses have revealed that
presenilin is a modulator and mandatory component of the γ-secretase–mediated cleavage of APP.
Genetic studies have identified three other components required for γ-secretase activity:
nicastrin,
anterior pharynx defective 1 and
presenilin enhancer 2.
By coexpression of presenilin with the other three components, the authors managed to
reconstitute γ-secretase activity.
Tomita and Iwatsubo determined using the substituted cysteine accessibility method and by topological analyses, that
the catalytic aspartates are located at the center of the nine transmembrane domains of presenilin,
by revealing the exact location of the enzyme’s catalytic site.
The minireview also describes in detail the formerly enigmatic mechanism of γ-secretase mediated cleavage.
SPP, an enzyme that cleaves remnant signal peptides in the membrane
during the biogenesis of membrane proteins and
signal peptides from major histocompatibility complex type I,
also is involved in the maturation of proteins of the hepatitis C virus and GB virus B.
Bioinformatics methods have revealed in fruit flies and mammals four SPP-like proteins,
two of which are involved in immunological processes.
By using γ-secretase inhibitors and modulators, it has been confirmed
that SPP shares a similar GxGD active site and proteolytic activity with γ-secretase.
Upon purification of the human SPP protein with the baculovirus/Sf9 cell system,
single-particle analysis revealed further structural and functional details.
HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection
Experimental and computational evidence suggests that
HLAs preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that
this preference may provide improved clearance of infection in several viral systems.
To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study
performed during the 2009–2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with
A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,
were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
are common in certain indigenous populations in which increased pH1N1 morbidity has been reported.
HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection.
The computational tools used in this study may be useful predictors of potential morbidity and
identify immunologic differences of new variant influenza strains
more accurately than evolutionary sequence comparisons.
Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases
might advance clinical practices for severe H1N1 infections among genetically susceptible populations.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ.
Cold Spring Harbor Symposia on Quantitative Biology 11/2011; 76:235-46. http://dx.doi.org:/10.1101/sqb.2011.76.010694
Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes,
metabolomics can point to potential new sites for therapeutic intervention.
Particularly promising enzymatic targets are those that
carry increased flux in the disease state.
Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for
finding new drug targets using metabolomics and isotope tracers.
The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus,
metabolomic analysis of infected versus mock-infected cells revealed
dramatic concentration changes around the current antiviral target enzymes.
Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes
in a region of metabolism unrelated to the current antiviral target.
Instead, it pointed to the tricarboxylic acid (TCA) cycle and
its efflux to feed fatty acid biosynthesis as a potential preferred target.
Isotope tracer studies revealed that cytomegalovirus greatly increases flux through
the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase.
Inhibition of this enzyme blocks human cytomegalovirus replication.
Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of
metabolomics as a drug target discovery strategy will be
successful clinical development of therapeutics hitting these new targets.
Related References
Use of metabolic pathway flux information in targeted cancer drug design. Drug Discovery Today: Therapeutic Strategies 1:435-443, 2004.
Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics. 2005;5(5):293-302. Review. PMID: 16196499
Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.
Mini Rev Med Chem. 2005 Jan;5(1):13-20. Review. PMID: 15638788 [PubMed – indexed for MEDLINE] Related citations
Development of Tracer-Based Metabolomics and its Implications for the Pharmaceutical Industry. Int J Pharm Med 2007; 21 (3): 217-224.
Use of metabolic pathway flux information in anticancer drug design. Ernst Schering Found Symp Proc. 2007;(4):189-203. Review. PMID: 18811058
Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice. J Pharmacol Exp Ther. 2011 Jul;338(1):70-81. http://dx.doi.org:/10.1124/jpet.111.179986. PMID: 21471191
Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the
[U-C(6)]-d-glucose tracer in mice. Metabolomics. 2009 Sep;5(3):336-345. PMID: 19718458
Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. FASEB J. 2013 Jul;27(7):2845-54. http://dx.doi.org:/10.1096/fj.12-216929. PMID: 23515442
Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery
The change in drug discovery strategy from ‘classical’ function-first approaches (in which the assay of drug function was at the tissue or organism level),
with mechanistic studies potentially coming later, to more-recent target-based approaches where initial assays usually involve assessing the interactions
of drugs with specified (and often cloned, recombinant) proteins in vitro. In the latter cases, effects in vivo are assessed later, with concomitantly high levels of attrition.
Arguably the two chief hallmarks of the systems biology approach are:
(i) that we seek to make mathematical models of our systems iteratively or in parallel with well-designed ‘wet’ experiments, and
(ii) that we do not necessarily start with a hypothesis but measure as many things as possible (the ’omes) and
let the data tell us the hypothesis that best fits and describes them.
Although metabolism was once seen as something of a Cinderella subject,
there are fundamental reasons to do with the organisation of biochemical networks as
to why the metabol(om)ic level – now in fact seen as the ‘apogee’ of the ’omics trilogy –
is indeed likely to be far more discriminating than are
changes in the transcriptome or proteome.
The next two subsections deal with these points and Fig. 2 summarises the paper in the form of a Mind Map.
Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated. Continuing
unmet medical needs remain a major driver for new research.
Drug discovery approaches in this field have mirrored industry trends, leading to a recent
increase in the number of molecules entering development.
However, worrisome trends and newer hurdles are also apparent. The history of two newer drug classes—
glucagon-like peptide-1 receptor agonists and
dipeptidyl peptidase-4 inhibitors—
illustrates both progress and challenges. Future success requires that researchers learn from these experiences and
continue to explore and apply new technology platforms and research paradigms.
The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts an even greater diabetes burden (>430 million people afflicted) by 2030, which will disproportionately affect developing nations (International Diabetes Federation, 2011). Yet
existing drug classes for diabetes, obesity, and comorbid cardiovascular (CV) conditions have substantial limitations.
Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable shortcomings. In general,
Therefore, clinicians must often use combination therapy, adding additional agents over time. Ultimately many patients will need to use insulin—a therapeutic class first introduced in 1922. Most existing agents also have
issues around safety and tolerability as well as dosing convenience (which can impact patient compliance).
Pharmacometabolomics, also known as pharmacometabonomics, is a field which stems from metabolomics,
the quantification and analysis of metabolites produced by the body.
It refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to
This concludes a long step-by-step journey into rediscovering biological processes from the genome as a framework to the remodeled and reconstituted cell through a number of posttranscription and posttranslation processes that modify the proteome and determine the metabolome. The remodeling process continues over a lifetime. The process requires a balance between nutrient intake, energy utilization for work in the lean body mass, energy reserves, endocrine, paracrine and autocrine mechanisms, and autophagy. It is true when we look at this in its full scope – What a creature is man?
Smith CA, O’Maille G, Want EJ, Qin C, Trauger SA, Brandon TR, Custodio DE, Abagyan R, Siuzdak G METLIN: A Metabolite Mass Spectral Database Therapeutic Drug Monitoring, 2005, 27(6), 747-751
Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”, the study of their small-molecule metabolite profiles.[1] The metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes.[2]mRNAgene expression data and proteomic analyses reveal the set of gene products being produced in the cell, data that represents one aspect of cellular function. Conversely, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology and functional genomics is to integrate proteomic, transcriptomic, and metabolomic information to provide a better understanding of cellular biology.
The term “metabolic profile” was introduced by Horning, et al. in 1971 after they demonstrated that gas chromatography-mass spectrometry (GC-MS) could be used to measure compounds present in human urine and tissue extracts. The Horning group, along with that of Linus Pauling and Arthur B. Robinson led the development of GC-MS methods to monitor the metabolites present in urine through the 1970s.
Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples.This first study on muscle highlighted the value of NMR in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic angle spinning, NMR continues to be a leading analytical tool to investigate metabolism. Efforts to utilize NMR for metabolomics have been influenced by the laboratory of Dr. Jeremy Nicholson at Birkbeck College, University of London and later at Imperial College London. In 1984, Nicholson showed 1H NMR spectroscopy could potentially be used to diagnose diabetes mellitus, and later pioneered the application of pattern recognition methods to NMR spectroscopic data.
In 2005, the first metabolomics web database, METLIN, for characterizing human metabolites was developed in the Siuzdak laboratory at The Scripps Research Institute and contained over 10,000 metabolites and tandem mass spectral data. As of September 2012, METLIN contains over 60,000 metabolites as well as the largest repository of tandem mass spectrometry data in metabolomics.
On 23 January 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components. Similar projects have been underway in several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years.
As late as mid-2010, metabolomics was still considered an “emerging field”. Further, it was noted that further progress in the field depended in large part, through addressing otherwise “irresolvable technical challenges”, by technical evolution of mass spectrometry instrumentation.
Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. The word was coined in analogy with transcriptomics and proteomics; like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method. The first metabolite database(called METLIN) for searching m/z values from mass spectrometry data was developed by scientists at The Scripps Research Institute in 2005. In January 2007, scientists at the University of Alberta and the University of Calgary completed the first draft of the human metabolome. They catalogued approximately 2500 metabolites, 1200 drugs and 3500 food components that can be found in the human body, as reported in the literature. This information, available at the Human Metabolome Database (www.hmdb.ca) and based on analysis of information available in the current scientific literature, is far from complete.
Each type of cell and tissue has a unique metabolic ‘fingerprint’ that can elucidate organ or tissue-specific information, while the study of biofluids can give more generalized though less specialized information. Commonly used biofluids are urine and plasma, as they can be obtained non-invasively or relatively non-invasively, respectively. The ease of collection facilitates high temporal resolution, and because they are always at dynamic equilibrium with the body, they can describe the host as a whole.
Metabolites are the intermediates and products of metabolism. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size.
A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes. By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous (produced by the host organism) or exogenous. Metabolites of foreign substances such as drugs are termed xenometabolites. The metabolome forms a large network of metabolic reactions, where outputs from one enzymaticchemical reaction are inputs to other chemical reactions.
Metabonomics is defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification”. The word origin is from the Greek μεταβολή meaning change and nomos meaning a rule set or set of laws. This approach was pioneered by Jeremy Nicholson at Imperial College London and has been used in toxicology, disease diagnosis and a number of other fields. Historically, the metabonomics approach was one of the first methods to apply the scope of systems biology to studies of metabolism.
There is a growing consensus that ‘metabolomics’ places a greater emphasis on metabolic profiling at a cellular or organ level and is primarily concerned with normal endogenous metabolism. ‘Metabonomics’ extends metabolic profiling to include information about perturbations of metabolism caused by environmental factors (including diet and toxins), disease processes, and the involvement of extragenomic influences, such as gut microflora. This is not a trivial difference; metabolomic studies should, by definition, exclude metabolic contributions from extragenomic sources, because these are external to the system being studied.
Toxicity assessment/toxicology. Metabolic profiling (especially of urine or blood plasma samples) detects the physiological changes caused by toxic insult of a chemical (or mixture of chemicals).
Functional genomics. Metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself—for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes.
Nutrigenomics is a generalised term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non- nutrients.
The problem with genomics was it was set as explanation for everything. In fact, when something is genetic in nature the genomic reasoning works fine. However, this means whenever an inborn error is found and only in this case the genomic knowledge afterwards may indicate what is wrong and not the completely way to put biology upside down by reading everything in the DNA genetic as well as non-genetic problems.
Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012
conformational changes leading to substrate efflux.img
The cellular response is defined by a network of chemogenomic response signatures.
Humans, mammals, plants and animals, and eukaryotes and prokaryotes all share a common denominator in their manner of existence. It makes no difference whether they inhabit the land, or the sea, or another living host. They exist by virtue of their metabolic adaptation by way of taking in nutrients as fuel, and converting the nutrients to waste in the expenditure of carrying out the functions of motility, breakdown and utilization of fuel, and replication of their functional mass.
There are essentially two major sources of fuel, mainly, carbohydrate and fat. A third source, amino acids which requires protein breakdown, is utilized to a limited extent as needed from conversion of gluconeogenic amino acids for entry into the carbohydrate pathway. Amino acids follow specific metabolic pathways related to protein synthesis and cell renewal tied to genomic expression.
Carbohydrates are a major fuel utilized by way of either of two pathways. They are a source of readily available fuel that is accessible either from breakdown of disaccharides or from hepatic glycogenolysis by way of the Cori cycle. Fat derived energy is a high energy source that is metabolized by one carbon transfers using the oxidation of fatty acids in mitochondria. In the case of fats, the advantage of high energy is conferred by chain length.
Carbohydrate metabolism has either of two routes of utilization. This introduces an innovation by way of the mitochondrion or its equivalent, for the process of respiration, or aerobic metabolism through the tricarboxylic acid, or Krebs cycle. In the presence of low oxygen supply, carbohydrate is metabolized anaerobically, the six carbon glucose being split into two three carbon intermediates, which are finally converted from pyruvate to lactate. In the presence of oxygen, the lactate is channeled back into respiration, or mitochondrial oxidation, referred to as oxidative phosphorylation. The actual mechanism of this process was of considerable debate for some years until it was resolved that the mechanism involve hydrogen transfers along the “electron transport chain” on the inner membrane of the mitochondrion, and it was tied to the formation of ATP from ADP linked to the so called “active acetate” in Acetyl-Coenzyme A, discovered by Fritz Lipmann (and Nathan O. Kaplan) at Massachusetts General Hospital. Kaplan then joined with Sidney Colowick at the McCollum Pratt Institute at Johns Hopkins, where they shared tn the seminal discovery of the “pyridine nucleotide transhydrogenases” with Elizabeth Neufeld, who later established her reputation in the mucopolysaccharidoses (MPS) with L-iduronidase and lysosomal storage disease.
This chapter covers primarily the metabolic pathways for glucose, anaerobic and by mitochondrial oxidation, the electron transport chain, fatty acid oxidation, galactose assimilation, and the hexose monophosphate shunt, essential for the generation of NADPH. The is to be more elaboration on lipids and coverage of transcription, involving amino acids and RNA in other chapters.
The subchapters are as follows:
1.1 Carbohydrate Metabolism
1.2 Studies of Respiration Lead to Acetyl CoA
1.3 Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief
1.4 The Multi-step Transfer of Phosphate Bond and Hydrogen Exchange Energy
This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases. It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time bachalaureate degree reader in the biological sciences. Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career.
In the Preface, I failed to disclose that the term Metabolomics applies to plants, animals, bacteria, and both prokaryotes and eukaryotes. The metabolome for each organism is unique, but from an evolutionary perspective has metabolic pathways in common, and expressed in concert with the environment that these living creatures exist. The metabolome of each has adaptive accommodation with suppression and activation of pathways that are functional and necessary in balance, for its existence. Was it William Faulkner who said in his Nobel Prize acceptance that mankind shall not merely exist, but survive? That seems to be the overlying theme for all of life. If life cannot persist, a surviving “remnant” might continue. The history of life may well be etched into the genetic code, some of which is not expressed.
This work is apportioned into chapters in a sequence that is first directed at the major sources for the energy and the structure of life, in the carbohydrates, lipids, and fats, which are sourced from both plants and animals, and depending on their balance, results in an equilibrium, and a disequilibrium we refer to as disease. There is also a need to consider the nonorganic essentials which are derived from the soil, from water, and from the energy of the sun and the air we breathe, or in the case of water-bound metabolomes, dissolved gases.
In addition to the basic essential nutrients and their metabolic utilization, they are under cellular metabolic regulation that is tied to signaling pathways. In addition, the genetic expression of the organism is under regulatory control by the interaction of RNAs that interact with the chromatin genetic framework, with exosomes, and with protein modulators.This is referred to as epigenetics, but there are also drivers of metabolism that are shaped by the interactions between enzymes and substartes, and are related to the tertiary structure of a protein. The framework for diseases in a separate chapter. Pharmaceutical interventions that are designed to modulate specific metabolic targets are addressed as the pathways are unfolded. Neutraceuticals and plant based nutrition are covered in Chapter 8.
Chapter 1: Metabolic Pathways
Chapter 2. Lipid Metabolism
Chapter 3. Cell Signaling
Chapter 4. Protein Synthesis and Degradation
Chapter 5: Sub-cellular Structure
Chapter 6: Proteomics
Chapter 7: Metabolomics
Chapter 8. Impairments in Pathological States: Endocrine Disorders; Stress Hypermetabolism and Cancer
Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
Reviewer and Curator: Larry H. Bernstein, MD, FCAP
The human genome is estimated to encode over 30,000 genes, and to be responsible for generating more than 100,000 functionally distinct proteins. Understanding the interrelationships among
genes,
gene products, and
dietary habits
is fundamental to identifying those who will benefit most from or be placed at risk by intervention strategies.
Unraveling the multitude of
nutrigenomic,
proteomic, and
metabolomic patterns
that arise from the ingestion of foods or their
bioactive food components
will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as
microarrays,
RNA interference, and
nanotechnologies,
will provide needed insights into molecular targets for specific bioactive food components and
how they harmonize to influence individual phenotypes(1).
Nutrigenetics asks the question how individual genetic disposition, manifesting as
single nucleotide polymorphisms,
copy-number polymorphisms and
epigenetic phenomena,
affects susceptibility to diet.
Nutrigenomics addresses the inverse relationship, that is how diet influences
gene transcription,
protein expression and
metabolism.
A major methodological challenge and first pre-requisite of nutrigenomics is integrating
genomics (gene analysis),
transcriptomics (gene expression analysis),
proteomics (protein expression analysis) and
metabonomics (metabolite profiling)
to define a “healthy” phenotype. The long-term deliverable of nutrigenomics is personalised nutrition (2).
Science is beginning to understand how genetic variation and epigenetic events
alter requirements for, and responses to, nutrients (nutrigenomics).
At the same time, methods for profiling almost all of the products of metabolism in a single sample of blood or urine are being developed (metabolomics). Relations between
diet and nutrigenomic and metabolomic profiles and
between those profiles and health
have become important components of research that could change clinical practice in nutrition.
Most nutrition studies assume that all persons have average dietary requirements, and the studies often
do not plan for a large subset of subjects who differ in requirements for a nutrient.
Large variances in responses that occur when such a population exists
can result in statistical analyses that argue for a null effect.
If nutrition studies could better identify responders and differentiate them from nonresponders on the basis of nutrigenomic or metabolomic profiles,
the sensitivity to detect differences between groups could be greatly increased, and
the resulting dietary recommendations could be appropriately targeted (3).
In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. Following this trend,
Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that
aims to elucidate how diet can influence human health.
It is already well known that bioactive food compounds can interact with genes affecting
transcription factors,
protein expression and
metabolite production.
The study of these complex interactions requires the development of
advanced analytical approaches combined with bioinformatics.
Thus, to carry out these studies
Transcriptomics,
Proteomics and
Metabolomics
approaches are employed together with an adequate integration of the information that they provide(4).
Metabonomics is a diagnostic tool for metabolic classification of individuals with the asset of quantitative, non-invasive analysis of easily accessible human body fluids such as urine, blood and saliva. This feature also applies to some extent to Proteomics, with the constraint that
the latter discipline is more complex in terms of composition and dynamic range of the sample.
Apart from addressing the most complex “Ome”, Proteomics represents
the only platform that delivers not only markers for disposition and efficacy
but also targets of intervention.
Application of integrated Omic technologies will drive the understanding of
interrelated pathways in healthy and pathological conditions and
will help to define molecular ‘switchboards’,
necessary to develop disease related biomarkers.
This will contribute to the development of new preventive and therapeutic strategies for both pharmacological and nutritional interventions (5).
Human health is affected by many factors. Diet and inherited genes play an important role. Food constituents,
including secondary metabolites of fruits and vegetables, may
interact directly with DNA via methylation and changes in expression profiles (mRNA, proteins)
which results in metabolite content changes.
Many studies have shown that
food constituents may affect human health and
the exact knowledge of genotypes and food constituent interactions with
both genes and proteins may delay or prevent the onset of diseases.
Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research, namely in the field of genomics and transcriptomics, exist. Studies on epigenetics and RNome significance have been launched. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of
comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids (6).
New experimental tools that investigate gene function at the subcellular, cellular, organ, organismal, and ecosystem level need to be developed. New bioinformatics tools to analyze and extract meaning
from increasingly systems-based datasets will need to be developed.
These will require, in part, creation of entirely new tools. An important and revolutionary aspect of “The 2010 Project” is that it implicitly endorses
the allocation of resources to attempts to assign function to genes that have no known function.
This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that
for the first time it is feasible to envision a whole-systems approach to gene and protein function.
This whole-systems approach promises to be orders of magnitude more efficient than the conventional approach (7).
The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy (8). Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of
(a) genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,
(b) individualized medical nutrition therapy for disease management, and
(c) better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that
maximize benefit and minimize adverse outcomes within genetically diverse human populations.
As the field of nutritional genomics matures, which will include filling fundamental gaps in
knowledge of nutrient-genome interactions in health and disease and
demonstrating the potential benefits of customizing nutrition prescriptions based on genetics,
registered dietitians will be faced with the opportunity of making genetically driven dietary recommendations aimed at improving human health.
The new era of nutrition research translates empirical knowledge to evidence-based molecular science (9). Modern nutrition research focuses on
promoting health,
preventing or delaying the onset of disease,
optimizing performance, and
assessing risk.
Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics
build the science foundation for understanding human variability in
preferences, requirements, and responses to diet and
may become the future tools for consumer assessment
motivated by personalized nutritional counseling for health maintenance and disease prevention.
The primary aim of ―omic‖ technologies is
the non-targeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample.
By their nature, these technologies reveal unexpected properties of biological systems.
A second and more challenging aspect of ―omic‖ technologies is
the refined analysis of quantitative dynamics in biological systems (10).
For metabolomics, gas and liquid chromatography coupled to mass spectrometryare well suited for coping with
high sample numbers in reliable measurement times with respect to
both technical accuracy and the identification and quantitation of small-molecular-weight metabolites.
This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology.
In modern nutrition research, mass spectrometry has developed into a tool
to assess health, sensory as well as quality and safety aspects of food.
In this review, we focus on health-related benefits of food components and, accordingly,
on biomarkers of exposure (bioavailability) and bioefficacy.
Current nutrition research focuses on unraveling the link between
dietary patterns,
individual foods or
food constituents and
the physiological effects at cellular, tissue and whole body level
after acute and chronic uptake.
The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand
the impact of food on defined health outcomes.
Both strongly depend on appropriate analytical tools
to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and
to monitor molecular changes in the body in a highly specific and sensitive manner.
Based on these requirements,
mass spectrometry has become the analytical method of choice
with broad applications throughout all areas of nutrition research (11).
Recent advances in high data-density analytical techniques offer unrivaled promise for improved medical diagnostics in the coming decade. Genomics, proteomics and metabonomics (as well as a whole slew of less well known ―omics‖ technologies) provide a detailed descriptor of each individual. Relating the large quantity of data on many different individuals to their current (and possibly even future) phenotype is a task not well suited to classical multivariate statistics. The datasets generated by ―omics‖ techniques very often violate the requirements for multiple regression. However, another statistical approach exists, which is already well established in areas such as medicinal chemistry and process control, but which is new to medical diagnostics, that can overcome these problems. This approach, called megavariate analysis (MVA),
has the potential to revolutionise medical diagnostics in a broad range of diseases.
It opens up the possibility of expert systems that can diagnose the presence of many different diseases simultaneously, and
even make exacting predictions about the future diseases an individual is likely to suffer from (12).
Cardiovascular diseases
Cardiovascular diseases are the leading cause of morbidity and mortality in Western countries. Although coronary thrombosis is the final event in acute coronary syndromes,
there is increasing evidence that inflammation also plays a role in development of atherosclerosis and its clinical manifestations, such as
myocardial infarction, stroke, and peripheral vascular disease.
The beneficial cardiovascular health effects of
diets rich in fruits and vegetables are in part mediated by their flavanol content.
This concept is supported by findings from small-scale intervention studies with surrogate endpoints including
endothelium-dependent vasodilation,
blood pressure,
platelet function, and
glucose tolerance.
Mechanistically, short term effects on endothelium-dependent vasodilation
following the consumption of flavanol-rich foods, as well as purified flavanols,
have been linked to an increased nitric oxide bioactivity.
The critical biological target(s) for flavanols have yet to be identified (13), but we are beginning to see over the horizon.
Nutritional sciences
Nutrition sciences apply
transcriptomics,
proteomics and
metabolomics
to molecularly assess nutritional adaptations.
Transcriptomics can generate a
holistic overview on molecular changes to dietary interventions.
Proteomics is most challenging because of the higher complexity of proteomes as compared to transcriptomes and metabolomes. However, it delivers
not only markers but also
targets of intervention, such as
enzymes or transporters, and
it is the platform of choice for discovering bioactive food proteins and peptides.
Metabolomics is a tool for metabolic characterization of individuals and
can deliver metabolic endpoints possibly related to health or disease.
Omics in nutrition should be deployed in an integrated fashion to elucidate biomarkers
for defining an individual’s susceptibility to diet in nutritional interventions and
for assessing food ingredient efficacy (14).
The more elaborate tools offered by metabolomics opened the door to exploring an active role played by adipose tissue that is affected by diet, race, sex, and probably age and activity. When the multifactorial is brought into play, and the effect of changes in diet and activities studied we leave the study of metabolomics and enter the world of ―metabonomics‖. Adiponectin and adipokines arrive (15-22). We shall discuss ―adiposity‖ later.
Potential Applications of Metabolomics
Either individually or grouped as a profile, metabolites are detected by either
nuclear magnetic resonance spectroscopy or mass spectrometry.
There is potential for a multitude of uses of metabolome research, including
the early detection and diagnosis of cancer and as
both a predictive and pharmacodynamic marker of drug effect.
However, the knowledge regarding metabolomics, its technical challenges, and clinical applications is unappreciated
even though when used as a translational research tool,
it can provide a link between the laboratory and clinic.
Precise numbers of human metabolites is unknown, with estimates ranging from the thousands to tens of thousands. Metabolomics is a term that encompasses several types of analyses, including
(a) metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;
(b) metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and
(c) target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing
only a few selected metabolites that comprise a specific biochemical pathway.
Dynamic Construct of the –Omics
Dynamic Construct of the –Omics
Iron metabolism – Anemia
Hepcidin is a key hormone governing mammalian iron homeostasis and may be directly or indirectly involved in the development of most iron deficiency/overload and inflammation-induced anemia. The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and hepcidin regulation. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. While patients present with both ACD and iron deficiency anemia (ACD/IDA), the latter results from chronic blood loss. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In ACD, serum hepcidin concentrations are elevated, which is related to reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects. ACD/IDA patients, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Hepcidin elevation may affect iron transport in ACD and ACD/IDA and it is more responsive to iron demand with IDA than to inflammation. Hepcidin determination may aid in selecting appropriate therapy for these patients (23).
There is correlation between serum hepcidin, iron and inflammatory indicators associated with anemia of chronic disease (ACD), ACD, ACD concomitant iron-deficiency anemia (ACD/IDA), pure IDA and acute inflammation (AcI) patients. Hepcidin levels in anemia types were statistically different, from high to low: ACD, AcI > ACD/IDA > the control > IDA. Serum ferritin levels were significantly increased in ACD and AcI patients but were decreased significantly in ACD/IDA and IDA. Elevated serum EPO concentrations were found in ACD, ACD/IDA and IDA patients but not in AcI patients and the controls. A positive correlation exists between hepcidin and IL-6 levels only in ACD/IDA, AcI and the control groups. A positive correlation between hepcidin and ferritin was marked in the control group, while a negative correlation between hepcidin and ferritin was noted in IDA. The significant negative correlation between hepcidin expression and reticulocyte count was marked in both ACD/IDA and IDA groups. If the hepcidin role in pathogenesis of ACD, ACD/IDA and IDA, it could be a potential marker for detection and differentiation of these anemias (24).
Cancer
Because cancer cells are known to possess a highly unique metabolic phenotype, development of specific biomarkers in oncology is possible and might be used in identifying fingerprints, profiles, or signatures to detect the presence of cancer, determine prognosis, and/or assess the pharmacodynamic effects of therapy (25).
HDM2, a negative regulator of the tumor suppressor p53, is over-expressed in many cancers that retain wild-type p53. Consequently, the effectiveness of chemotherapies that induce p53 might be limited, and inhibitors of the HDM2–p53 interaction are being sought as tumor-selective drugs. A binding site within HDM2 has been dentified which can be blocked with peptides inducing p53 transcriptional activity. A recent report demonstrates the principle using drug-like small molecules that target HDM2 (26).
Obesity, CRP, interleukins, and chronic inflammatory disease
Elevated CRP levels and clinically raised CRP levels were present in 27.6% and 6.7% of the population, respectively. Both overweight (body mass index [BMI], 25-29.9 kg/m2) and obese (BMI, 30 kg/m2) persons were more likely to have elevated CRP levels than their normal-weight counterparts (BMI, <25 kg/m2). After adjusting for potential confounders, the odds ratio (OR) for elevated CRP was 2.13 for obese men and 6.21 for obese women. In addition, BMI was associated with clinically raised CRP levels in women, with an OR of 4.76 (95% CI, 3.42-6.61) for obese women. Waist-to-hip ratio was positively associated with both elevated and clinically raised CRP levels, independent of BMI. Restricting the analyses to young adults (aged 17-39 years) and excluding smokers, persons with inflammatory disease, cardiovascular disease, or diabetes mellitus and estrogen users did not change the main findings (27).
A study of C-reactive protein and interleukin-6 with measures of obesity and of chronic infection as their putative determinants related levels of C-reactive protein and interleukin-6 to markers of the insulin resistance syndrome and of endothelial dysfunction. Levels of C-reactive protein were significantly related to those of interleukin-6 (r=0.37, P<0.0005) and tumor necrosis factor-a (r=0.46, P<0.0001), and concentrations of C-reactive protein were related to insulin resistance as calculated from the homoeostasis model and to markers of endothelial dysfunction (plasma levels of von Willebrand factor, tissue plasminogen activator, and cellular fibronectin). A mean standard deviation score of levels of acute phase markers correlated closely with a similar score of insulin resistance syndrome variables (r=0.59, P<0.00005) and the data suggested that adipose tissue is an important determinant of a low level, chronic inflammatory state as reflected by levels of interleukin-6, tumor necrosis factor-a, and C-reactive protein (28).
A number of other studies have indicated the inflammatory ties of visceral obesity to adipose tissue metabolic profiles, suggesting a role in ―metabolic syndrome‖. There is now a concept of altered liver metabolism in ―non-alcoholic‖ fatty liver disease (NAFLD) progressing from steatosis to steatohepatitis (NASH) (31,32).
These unifying concepts were incomprehensible 50 years ago. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 16 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.
For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative G of phosphate hydrolysis than ATP.
The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:
(b) PEP Carboxykinase (Gluconeogenesis) catalyzes:
oxaloacetate + GTP — phosphoenolpyruvate + GDP + CO2
The concept of anomalies in the pathways with respect to diabetes was sketchy then, and there was much to be filled in. This has been substantially done, and is by no means complete. However, one can see how this comes into play with diabetic ketoacidosis accompanied by gluconeogenesis and in severe injury or sepsis with peripheral proteolysis to provide gluconeogenic precursors. The reprioritization of liver synthetic processes is also brought into play with the conundrum of protein-energy malnutrition.
The picture began to be filled in with the improvements in technology that emerged at the end of the 1980s with the ability to profile tissue and body fluids by NMR and by MS. There was already a good inkling of a relationship of type 2 diabetes to major indicators of CVD (29,30). And a long suspected relationship between obesity and type 2 diabetes was evident. But how did it tie together?
End Stage Renal Disease and Cardiovascular Risk
Mortality is markedly elevated in patients with end-stage renal disease. The leading cause of death is cardiovascular disease.
As renal function declines,
the prevalence of both malnutrition and cardiovascular disease increase.
Malnutrition and vascular disease correlate with the levels of
markers of inflammation in patients treated with dialysis and in those not yet on dialysis.
The causes of inflammation are likely to be multifactorial. CRP levels are associated with cardio-vascular risk in the general population.
The changes in endothelial cell function,
in plasma proteins, and
in lpiids in inflammation
are likely to be atherogenic.
That cardiovascular risk is inversely correlated with serum cholesterol in dialysis patients, suggests that
hyperlipidemia plays a minor role in the incidence of cardiovascular disease.
Hypoalbuminemia, ascribed to malnutrition, has been one of the most powerful risk factors that predict all-cause and cardiovascular mortality in dialysis patients. The presence of inflammation, as evidenced by increased levels of specific cytokines (interleukin-6 and tumor necrosis factor a) or acute-phase proteins (C-reactive protein and serum amyloid A), however, has been found to be associated with vascular disease in the general population as well as in dialysis patients. Patients have
loss of muscle mass and changes in plasma composition—decreases in serum albumin, prealbumin, and transferrin levels, also associated with malnutrition.
Inflammation alters
lipoprotein structure and function as well as
endothelial structure and function
to favor atherogenesis and increases
the concentration of atherogenic proteins in serum.
In addition, proinflammatory compounds, such as
advanced glycation end products, accumulate in renal failure, and
defense mechanisms against oxidative injury are reduced,
contributing to inflammation and to its effect on the vascular endothelium (33,34).
Endogenous copper can play an important role in postischemic reperfusion injury, a condition associated with endothelial cell activation and increased interleukin 8 (IL-8) production. Excessive endothelial IL-8 secreted during trauma, major surgery, and sepsis may contribute to the development of systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), and multiple organ failure (MOF). No previous reports have indicated that copper has a direct role in stimulating human endothelial IL-8 secretion. Copper did not stimulate secretion of other cytokines. Cu(II) appeared to be the primary copper ion responsible for the observed increase in IL-8 because a specific high-affinity Cu(II)-binding peptide, d-Asp-d-Ala-d-Hisd-Lys (d-DAHK), completely abolished this effect in a dose-dependent manner. These results suggest that Cu(II) may induce endothelial IL-8 by a mechanism independent of known Cu(I) generation of reactive oxygen species (35).
Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation. Activation of PAR_1 by thrombin and of PAR_2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Other receptors that can modulate responses of the cells activated by proteinases through PAR receptors are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa , essential to the inflammation and blood clotting processes (36).
The understanding of regulation of the inflammatory process in chronic inflammatory diseases is advancing.
Evidence consistently indicates that T-cells play a key role in initiating and perpetuating inflammation, not only via the production of soluble mediators but also via cell/cell contact interactions with a variety of cell types through membrane receptors and their ligands. Signalling through CD40 and CD40 ligand is a versatile pathway that is potently involved in all these processes. Many inflammatory genes relevant to atherosclerosis are influenced by the transcriptional regulator nuclear factor κ B (NFκB). In these events T-cells become activated by dendritic cells or inflammatory cytokines, and these T-cells activate, in turn, monocytes / macrophages, endothelial cells, smooth muscle cells and fibroblasts to produce pro-inflammatory cytokines, chemokines, the coagulation cascade in vivo, and finally matrix metalloproteinases, responsible for tissue destruction. Moreover, CD40 ligand at inflammatory sites stimulates fibroblasts and tissue monocyte/macrophage production of VEGF, leading to angiogenesis, which promotes and maintains the chronic inflammatory process.
NFκB plays a pivotal role in co-ordinating the expression of genes involved in the immune and inflammatory response, evoking tumor necrosis factor α (TNFα), chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8, matrix metalloproteinase enzymes (MMP), and genes involved in cell survival. A complex array of mechanisms, including T cell activation, leukocyte extravasation, tissue factor expression, MMP expression and activation, as well induction of cytokines and chemokines, implicated in atherosclerosis, are regulated by NFκB.
Expression of NFκB in the atherosclerotic milieu may have a number of potentially harmful consequences. IL-1 activates NFκB upregulating expression of MMP-1, -3, and -9. Oxidized LDL increases macrophage MMP-9, associated with increased nuclear binding of NFκB and AP-1. Expression of tissue factor, initiating the coagulation cascade, is regulated by NFκB. In atherosclerotic plaque cells, tissue factor antigen and activity were inhibited following over-expression of IκBα and dominant-negative IKK-2, but not by dominant negative IKK-1 or NIK. Tis supports the concept that activation of the ―canonical‖ pathway upregulates pro-thrombotic mediators involved in disease. Many of the cytokines and chemokines which have been detected in human atherosclerotic plaques are also regulated by NFκB. Over-expression of IκBα inhibits release of TNFα, IL-1, IL-6, and IL-8 in macrophages stimulated with LPS and CD40 ligand (CD40L). This report describes how NFκB activation upregulates major pro-inflammatory and pro-thrombotic mediators of atherosclerosis (37-41).
This review is both focused and comprehensive. The details of evolving methods are avoided in order to build the argument that a very rapid expansion of discovery has been evolving depicting disease, disease mechanisms, disease associations, metabolic biomarkers, study of effects of diet and diet modification, and opportunities for targeted drug development. The extent of future success will depend on the duration and strength of the developed interventions, and possibly the avoidance of dead end interventions that are unexpectedly bypassed. I anticipate the prospects for the interplay between genomics, metabolomics, metabonomics, and personalized medicine may be realized for several of the most common conditions worldwide within a few decades (42-44).
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Reporter and Curator: Larry H. Bernstein, MD, FCAP
This is fourth of a series of articles, lipid metabolism, that began with signaling and signaling pathways. These discussion lay the groundwork to proceed in later discussions that will take on a somewhat different approach. These are critical to develop a more complete point of view of life processes. I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different. The role of lipids in circulating plasma proteins as biomarkers for coronary vascular disease can be traced to the early work of Frederickson and the classification of lipid disorders. The very critical role of lipids in membrane structure in health and disease has had much less attention, despite the enormous importance, especially in the nervous system.
Signaling and signaling pathways
Signaling transduction tutorial.
Carbohydrate metabolism
3.1 Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence
Lipid metabolism
Protein synthesis and degradation
Subcellular structure
Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.
The major aspects of lipid metabolism are involved with
Fatty Acid Oxidationto produce energy or
the synthesis of lipids which is called Lipogenesis.
The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.
The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.
Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.
The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.
In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.
There are two major types of fatty acids – ω-3 and ω-6. There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured. Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.
Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.
The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.
In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.
A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.
Connections to Electron Transport and ATP:
One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:
Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP
In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.
Example with Palmitic Acid = 16 carbons = 8 acetyl groups
Number of turns of fatty acid spiral = 8-1 = 7 turns
ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.
This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.
NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP
Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain
Step
ATP produced
7
1
Step 4 (NAD+ to E.T.C.)
3
Step 6 (NAD+ to E.T.C.)
3
Step10 (NAD+ to E.T.C.)
3
Step 8 (FAD to E.T.C.)
2
NET
12 ATP
ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain
Step
ATP (used -) (produced +)
Step 1 (FAD to E.T.C.)
+2
Step 4 (NAD+ to E.T.C.)
+3
Total ATP
+5
7 turns
7 x 5 = 35
initial step
-1
NET
34 ATP
The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.
Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.
E.T.C = electron transport chain
Step
ATP produced
One acetyl CoA per turn C.A.C.
+12 ATP
8 Acetyl CoA = 8 turns C.A.C.
8 x 12 = 96 ATP
Fatty Acid Spiral
34 ATP
GRAND TOTAL
130 ATP
Fyodor Lynen
Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.
Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.
In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.
This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.
The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids
My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”
had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-
theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.
This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.
The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
the second was that the adenosine polyphosphate system plays a vital part in the cell,
not only in energy transfer, but
also in the regulation of the metabolic processes.
.
This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.
My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.
The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.
This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid
The answer provided by Martius was that citric acid is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).
It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.
In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that
fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.
As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.
This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.
My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,
we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.
Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.
In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.
I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme A.
Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.
Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.
All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.
Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.
SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver
Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1
1Department of Molecular Genetics, and 2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
J Clin Invest. 2002;109(9):1125–1131. http://dx.doi.org:/10.1172/JCI15593
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (1–4). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.
SREBPs: activation through proteolytic processing
SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.
In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).
Figure 1
Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1
Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.
SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.
When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.
SREBPs: two genes, three proteins
The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).
When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).
Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.
Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.
Knockout and transgenic mice
Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (9–16). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.
Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs
Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1–/– mice is due to the loss of the SREBP-1a transcript (16).
To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.
Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.
In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.
To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.
Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).
We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.
The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).
The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.
Transcriptional regulation of SREBP genes
Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).
The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).
Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (22–24). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.
LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.
SREBP-1c and the insulin/glucagon ratio
The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).
In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [3H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).
Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).
SREBPs in disease
Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).
Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.
The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).
Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).
In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because
humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,
it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.
SREBPs in liver: unanswered questions
The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.
Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.
Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.
Acknowledgments
Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.
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Membrane proteins rely on their interaction with membrane lipids to uphold its structure and maintain its functions as a protein. For membrane proteins to purify and crystallize, it is essential for the membrane protein to be in the appropriate lipid environment. Lipids assist in crystallization and stabilize the protein and provide lattice contacts. Lipids can also help obtain membrane protein structures in a native conformation. Membrane protein structures contain bound lipid molecules. Biological membranes are important in life, providing permeable barriers for cells and their organelles. The interaction between membrane proteins and lipids facilitates basic processes such as respiration, photosynthesis, transport, signal transduction and motility. These basic processes require a diverse group of proteins, which are encoded by 20-30% of an organism’s annotated genes.
There exist a great number of membrane lipids. Specifically, eukaryotic cells have a very complex collection of lipids that rely on many of the cell’s resources for its synthesis. Interactions between proteins and lipids can be very specific. Specific types of lipids can make a structure stable, provide control in insertion and folding processes, and help to assemble multisubunit complexes or supercomplexes, and most importantly, can significantly affect a membrane protein’s functions. Protein and lipid interactions are not sufficiently tight, meaning that lipids are retained during membrane protein purification. Since cellular membranes are fluid arrangements of lipids, some lipids affect interesting changes to membrane due to their characteristics. Glycosphigolipids and cholesterol tend to form small islands within the membranes, called lipid rafts, due to their physical properties. Some proteins also tend to cluster in lipid raft, while others avoid being in lipid rafts. However, the existence of lipid rafts in cells seems to be transitory.
Recent progress in determining membrane protein structure has brought attention to the importance of maintaining a favorable lipid environment so proteins to crystallize and purify successfully. Lipids assist in crystallization by stabilizing the protein fold and the relationships between subunits or monomers. The lipid content in protein-lipid detergent complexes can be altered by adjusting solubilisation and purification protocols, also by adding native or non-native lipids.
There are three type of membrane lipids: 1. Phospholipids: major class of membrane lipids. 2. glycolipids. 3. Cholesterols. Membrane lipids were started with eukaryotes and bacteria.
Lipids are often used as membrane constituents. The three major classes that membrane lipids are divided into are phospholipids, glycolipids, and cholesterol. Lipids are found in eukaryotes and bacteria. Although the lipids in archaea have many features that are related to the membrane formation that is similar with lipids of other organisms, they are still distinct from one another. The membranes of archaea differ in composition in three major ways. Firstly, the nonpolar chains are joined to a glycerol backbone by ether instead of esters, allowing for more resistance to hydrolysis. Second, the alkyl chains are not linear, but branched and make them more resistant to oxidation. The ability of archaeal lipids to resist hydrolysis and oxidation help these types of organisms to withstand the extreme conditions of high temperature, low pH, or high salt concentration. Lastly, the stereochemistry of the central glycerol is inverted. Membrane lipids have an extensive repertoire, but they possess a critical common structural theme in which they are amphipathic molecules, meaning they contain both a hydrophilic and hydrophobic moiety.
Membrane lipids are all closed bodies or boundaries separating substituent parts of the cell. The thickness of membranes is usually between 60 and 100 angstroms. These bodies are constructed from non-covalent assemblies. Their polar heads align with each other and their non-polar hydrocarbon tails align as well. The resulting stability is credited to hydrophobic interaction which proves to be quite stable due to the length of their hydrocarbon tails.
Membrane Lipids
Lipid Vesicles
Lipid vesicles, also known as liposomes, are vesicles that are essentially aqueous vesicles that are surrounded by a circular phospholipid bilayer. Like the other phospholipid structures, they have the hydrocarbon/hydrophobic tails facing inward, away from the aqueous solution, and the hydrophilic heads facing towards the aqueous solution. These vesicles are structures that form enclosed compartments of ions and solutes, and can be utilized to study the permeability of certain membranes, or to transfer these ions or solutes to certain cells found elsewhere.
Liposomes as vesicles can serve various clinical uses. Injecting liposomes containing medicine or DNA (for gene therapy) into patients is a possible method of drug delivery. The liposomes fuse with other cells’ membranes and therefore combine their contents with that of the patient’s cell. This method of drug delivery is less toxic than direct exposure because the liposomes carry the drug directly to cells without any unnecessary intermediate steps.
Because of the hydrophobic interactions among several phospholipids and glycolipids, a certain structure called the lipid bilayer or bimolecular sheet is favored. As mentioned earlier, phospholipids and glycolipids have both hydrophilic and hydrophobic moieties; thus, when several phospholipids or glycolipids come together in an aqueous solution, the hydrophobic tails interact with each other to form a hydrophobic center, while the hydrophilic heads interact with each other forming a hydrophilic coating on each side of the bilayer.
Effects of Omega-3 Fatty Acids on Lipids and Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis
This report is one of a group of evidence reports prepared by three Agency for Healthcare Research and Quality (AHRQ)-funded Evidence-Based Practice Centers (EPCs) on the role of omega-3 fatty acids (both from food sources and from dietary supplements) in the prevention or treatment of a variety of diseases. These reports were requested and funded by the Office of Dietary Supplements, National Institutes of Health. The three EPCs – the Southern California EPC (SCEPC, based at RAND), the Tufts-New England Medical Center (NEMC) EPC, and the University of Ottawa EPC – have each produced evidence reports. To ensure consistency of approach, the three EPCs collaborated on selected methodological elements, including literature search strategies, rating of evidence, and data table design.
The aim of these reports is to summarize the current evidence on the effects of omega-3 fatty acids on prevention and treatment of cardiovascular diseases, cancer, child and maternal health, eye health, gastrointestinal/renal diseases, asthma, immune- mediated diseases, tissue/organ transplantation, mental health, and neurological diseases and conditions. In addition to informing the research community and the public on the effects of omega-3 fatty acids on various health conditions, it is anticipated that the findings of the reports will also be used to help define the agenda for future research.
This report focuses on the effects of omega-3 fatty acids on immune- mediated diseases, bone metabolism, and gastrointestinal/renal diseases. Subsequent reports from the SCEPC will focus on cancer and neurological diseases and conditions.
This chapter provides a brief review of the current state of knowledge about the metabolism, physiological functions, and sources of omega-3 fatty acids.
The Recognition of Essential Fatty Acids
Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.
These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2
Fatty Acid Nomenclature
The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).
The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).
PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule. The trivial names, chemical names and abbreviations for the omega-3 fatty acids are detailed in Table 1.1. Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).
Fatty Acid Metabolism
Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.
No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.
Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).
The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.
The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.
Physiological Functions of EPA and AA
As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.
Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3
The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.
EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.
DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).
Dietary Sources and Requirements
Both ALA and LA are present in a variety of foods. LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA is present in some commonly used oils, including canola and soybean oil, and in some leafy green vegetables. Thus, the major dietary sources of ALA and LA are PUFA-rich vegetable oils. The proportion of LA to ALA as well as the proportion of those PUFAs to others varies considerably by the type of oil. With the exception of flaxseed, canola, and soybean oil, the ratio of LA to ALA in vegetable oils is at least 10 to 1. The ratios of LA to ALA for flaxseed, canola, and soy are approximately 1: 3.5, 2:1, and 8:1, respectively; however, flaxseed oil is not typically consumed in the North American diet. It is estimated that on average in the U.S., LA accounts for 89% of the total PUFAs consumed, and ALA accounts for 9%. Another estimate suggests that Americans consume 10 times more omega-6 than omega-3 fatty acids.4 Table 1.2 shows the proportion of omega 3 fatty acids for a number of foods.
Syntheis and Degradation
Source of Acetyl CoA for Fatty Acid Synthesis
step 1
ACP (acyl carrier protein)
synthesis requires acetyl CoA from citrate shuttle
conversion to fatty acyl co A in cytoplasm
ACP (acyl carrier protein)
FA synthesis not exactly reverse of catabolism
Fatty Acid Synthase
complete FA synthesis
Desaturation
Elongation and Desaturation of Fatty Acids
release of FAs from adiposites
Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2
ketone bodies
metabolism of ketone bodies
Arachidonoyl-mimicking
Arachidonate pathways
arachidonic acid derivatives
major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides
Model for the sterol-mediated proteolytic release of SREBPs from membrane
hormone regulation
insulin receptor and and insulin receptor signaling pathway (IRS)
islet brain glucose signaling
Fish source
omega FAs
Excessive omega 6s
omega 6s
diet and cancer
Patients at risk of FA deficiency
PPAR role
PPAR role
Omega 6_3 pathways
n3 vs n6 PUFAs
triene-teraene ratio
arachidonic acid, leukotrienes, PG and thromboxanes
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