Current Medicinal Chemistry - Immunology, Endocrine & Metabolic Agents - Volume 3, Issue 3, 2003

Years ago in graduate school I remember being told by my mentor to do a Folch extraction. As a young researcher I knew nothing of Folch or much about lipids for that matter. However, recently it occurred to me that perhaps Folch anticipated the recent genomics field and its related offspring-proteomics, metabolomics, and so on. In light of the recent explosion of “omics” of every variety and flavor, it would not be unreasonable to call Jordi Folch the father of “lipomics”. While focusing on brain biochemistry, his fundamental philosophy was that “everything must be accounted for” (1). This ultimately led to the 1957 paper entitled “A simple method for the isolation and purification of total lipids from animal tissues”; one of the most cited papers in all of life science literature (2). The paper describes the quantitative isolation of lipids from biological sources. At that time, researchers in the lipid field were not only focusing on the structure and mass of various lipid molecules, but also their biosynthesis. Konrad Bloch and Feodor Lynen were working out the biosynthetic details of cholesterol and fatty acid synthesis (3). In more recent times, research efforts have extended lipid biochemistry knowledge by focusing on regulatory mechanisms that control the biosynthesis and degradation of lipids. Short-term regulation involving changes in enzyme specific activity can be mediated by allosteric and covalent modification mechanisms while long-term regulation involving changes in protein levels can be mediated in part by altered gene expression. That abnormal regulation of lipid metabolism is associated with human diseases is undisputed; heart disease, obesity, dyslipidemia, and diabetes are only a few disorders that display various abnormalities in lipid metabolism. To treat those disorders, researchers continue to draw on past, basic metabolism work to guide drug discovery approaches and develop new therapeutics. Indeed, it was the intention of the contributors and me to put together a thematic issue of Current Medicinal Chemistry-Immunology, Endocrine, and Metabolic Agents that would integrate knowledge of short- and long-term regulatory mechanisms of key enzymes involved in lipid metabolism with practical aspects of discovering new drugs to treat diseases associated with lipid disorders. This issue represents that aim. This series of reviews is entitled “The Physiological and Pharmacological Regulation of Lipid Metabolism”. The first two articles provide up to date reviews on the regulatory mechanisms in the synthesis of acetyl-CoA, a central building block in lipid biosynthesis. Takahiro Fujino and co-workers nicely review the present state of knowledge on acetyl-CoA synthetases in an article entitled “Sources of acetyl-CoA: acetyl-CoA synthetase 1 and 2”. Next, Pieter Groot, Nigel Pearce, and Andrew Gribble describe an alternative pathway to generate acetyl-CoA, namely, through the enzyme ATP-citrate lyase. Their article is entitled, “ATP-citrate lyase: a potential target for hypolipidemic intervention.” These authors have weaved a wonderful story that integrates basic knowledge of lipid metabolism and short-term control mechanisms with a robust drug discovery approach to identify synthetic inhibitors of this enzyme. Once acetyl-CoA is formed it is can be utilized to synthesize cholesterol or fatty acids which involves HMG-CoA reductase and acetyl-CoA carboxylase, respectively; both enzymes are covered in this issue. Gene Ness provides an excellent overview of HMG-CoA reductase regulation with an emphasis on longterm control mechanisms elicited by hormones and the statin class of drugs in an article entitled, “Physiological and pharmacological regulation of hepatic 3-hydroxy-3-methylglutaryl Coenzyme A reductase”. With respect to acetyl-CoA carboxylase, Grover Waldrop and Jaqueline Stephens provide a review entitled “Targeting acetyl-CoA carboxylase for anti-obesity therapy”. Their novel approach to designing inhibitors of this enzyme provides an excellent example of utilizing knowledge of enzyme structure-function and substrates/products to synthesize “anti-metabolites”. The final article by Kathleen Knights is a thorough and fluid treatment of the fatty-acid CoA ligase family of enzymes. This family is central not only to lipid metabolism in general but particularly in the handling of xenobiotics that alter lipid metabolism. Indeed, the ligases catalyze the formation of xenobiotic acyl-CoA thioesters which actually may be mediators of some drug effects. The title of this review is “Long-chain fatty acid CoA ligases: the key to fatty acid activation, formation of xenobiotic acyl-CoA thioesters and lipophilic xenobiotic conjugates.” The contributors and I hope these series of articles spur novel ideas and approaches that may one day lead to new therapeutics to treat lipid and associated disorders. [1] Lees, M.B and Pope, A. Jordi Folch-Pi, In Biographical Memoirs, 2001, 79, pg 3-24. The National Acadamy Press, Washington, D.C. [2] Folch, J., Lees, M. and Stanley, G.H.S. J. Biol. Chem., 1957, 226, 497-509. [3] Kennedy, E.P. J. Biol. Chem., 2001, 276, 42619-42631.

Acetyl-CoA synthetase (AceCS) catalyzes the production of acetyl-CoA from acetate, CoA and ATP. There are two types of AceCS in mammals with different functions. One designated AceCS1 is a cytosolic enzyme expressed in the liver and plays a role in the production of acetyl-CoA for the synthesis of fatty acids and cholesterol. The other enzyme AceCS2 is a mitochondrial matrix enzyme that produces acetyl-CoAs mainly utilized for oxidation. Consistent with its function, the transcription of AceCS1 is regulated by SREBPs. In contrast, the expression of AceCS2 is upregulated during starvation and ketogenesis via unknown mechanisms. Specific inhibitors of AceCS may provide therapeutic agents for the treatment of obesity, cardiovascular diseases and type 2 diabetes.

Mammalian ATP-citrate lyase (EC 4.1.3.8) is the main enzyme responsible for the supply of acetyl-CoA for synthetic pathways. The enzyme is present in most tissues but particularly in those with an active de novo synthesis of fatty acids like adipose tissue and liver, especially during conditions of carbohydrate surplus. ATP-citrate lyase is the only enzyme shared by the synthetic pathways of fatty acid and cholesterol synthesis and due to this unique position, it has been proposed that inhibition of this enzyme may be more efficacious in correcting (mixed) hyperlipidemia than statins. In this manuscript we discuss the regulation of (hepatic) fatty acid and cholesterol synthesis and review the data on the in vitro and in vivo effects of inhibition of ATP-citrate lyase on lipid and lipoprotein metabolism. We also review the literature on the identification of inhibitors of ATP-citrate lyase. It is concluded that ATP-citrate lyase is a target for hypolipidemic intervention but that a final evaluation requires the identification of more potent inhibitors of this enzyme.

The conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate, catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), is considered to be the ratelimiting step in the overall pathway of cholesterol biosynthesis. Although expressed in virtually every tissue, HMGR is normally expressed at the highest level in liver. Also the magnitude of regulation of HMGR in liver exceeds that in any other tissue. Thus, this review focuses on regulation of hepatic HMGR. Feedback regulation by the end product of the pathway, cholesterol, has been, perhaps, the most extensively studied of the physiological and pharmacological factors that act to regulate HMGR expression. This constitutes an important homeostatic mechanism to maintain cholesterol levels within rather narrow limits. A series of recent studies have now demonstrated that this regulation occurs at the level of mRNA translation - not at the level of transcription as has been widely accepted. The rapid stimulation of hepatic HMGR activity by insulin is due to increased transcription rather than a change in phosphorylation status. Glucagon opposes this effect. Insulin's effect is mediated through phosphorylation of cAMP Response Element Binding Protein. Thyroid hormone acts, after a lag period of about 36 hrs, to markedly increase hepatic HMGR expression as a result of an increased rate of transcription as well as stabilization of the mRNA. Glucocorticoids oppose the mRNA stabilization. Statins act to increase hepatic HMGR protein levels as much as 700-fold through a combination of increased transcription, increased translation and stabilization of HMGR protein. Thyromimetics act to increase HMGR mRNA levels and also hepatic LDL receptor mRNA and protein levels. Oxylanosterols act to profoundly lower hepatic HMGR protein levels by acting to decrease the rate of translation. The physiological and pharmacological factors that modulate hepatic HMGR expression appear to employ a plethora of regulatory strategies. The molecular details of these regulatory mechanisms are the subjects of several current investigations.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long-chain fatty acids. ACC has three domains each having a different function in the conversion of acetyl CoA to malonyl CoA. The activity of mammalian ACC is regulated by allosteric effectors and by covalent modification, including phosphorylation and dephosphorylation by various hormones. Also, the regulation of ACC by phosphorylation is considered to be a target for hypolipidemic agents. There are two mammalian isoforms of ACC and recent studies suggest that genetic or pharmacological manipulation of both the mitochondrial isoform and cytosolic isoforms of ACC may be effective antiobesity or anti-diabetic treatments. The importance of ACC as an anti-obesity agent are supported by many studies which demonstrate that modulation of fatty acid synthesis with an inhibitor of fatty acid synthase can reduce food intake, increase fatty acid oxidation, and result in rapid and profound weight loss. This review focuses on recent reports that identify a new ACC inhibitor and suggest strategies for the development of new ACC inhibitors. We also present evidence to suggest that inhibition of ACC in non-adipose cells could be important and discuss recent studies which indicate that modulation of ACC may be useful in the treatment of some types of cancer. In summary, the pharmacological manipulation of acetyl-CoA carboxylase could be a suitable target as an anti-obesity agent and current evidence also suggests that inhibition of ACC could be a useful therapeutic for the treatment of both diabetes and cancer.

Long-chain-fatty-aid CoA ligases (EC 6.2.1.3) catalyse the bioactivation of fatty acids forming acyl-CoA thioesters that are then substrates for anabolic and catabolic pathways. In addition to these roles it is now recognised that fatty acyl-CoA esters are key regulatory molecules affecting numerous cellular systems and processes such as cell signalling, membrane fusion, protein acylation, protein kinase C activity, and gene transcription as natural ligands of the peroxisome proliferator-activated receptors. The key to both fatty acid activation and xenobiotic acyl-CoA formation is the role played by the long-chain ligases (LCL) that exist as a super family of membrane proteins. Focussing on information relevant to humans, multiplicity of LCLs, their structural features, and regulation are discussed. The fate of fatty acyl-CoAs and the role of LCL in directing fatty acyl-CoA traffic are also considered as these are integral to an appreciation of the consequences of xenobiotic-CoA conjugation and the formation of lipophilic conjugates. Although fatty acid activation is considered crucial to the provision of bioactive regulatory molecules, xenobiotic-CoA conjugation is at times a barely recognised route of drug metabolism. Knowledge of the consequences of xenobiotic-CoA formation and the pervasive intracellular role these conjugates play can provide further insight into xenobiotic metabolism and the interrelationship with lipid metabolism.

The nuclear receptors, LXRs and FXR, are important regulators of lipid homeostasis. Among their identified target genes is Apo E, which is a well-established plasma lipid transport protein. This lipoprotein has been extensively studied and a wide range of excellent reviews is available on it. Here, we reviewed the current status of Apo E regulation by LXRs and FXR, although a handful of publications are available on this topic. Functional response elements for LXRs and FXR have been identified in the Apo E / C-I / C-IV / C-II gene cluster. The emerging picture is that FXR regulates Apo E expression in the liver, whereas LXRs play a similar role in extrahepatic tissues. We further investigated the regulation of Apo E expression through LXR and FXR activation in liver- (HepG2), monocyte- (THP-1) and colon- (Caco-2) derived cell lines. Apo E transcript levels are increased in these three cell lines in response to LXR activation by the specific agonist T0901317. Upon FXR stimulation by GW4064, Apo E mRNA levels are increased in HepG2 and Caco-2, but not in the THP-1 cells, which do not express FXR. Interestingly, in the liver-derived HepG2 and colon-derived Caco-2 cell lines, which both express LXRs and FXR, Apo E expression can be controlled either by LXRs or FXR. In summary, these nuclear receptors display both distinct and overlapping functions in lipid metabolism. Apo E can be regulated by either LXRs or FXR, thus providing alternative mechanisms that control lipid homeostasis in different tissues.

In fact sepsis remains a major cause of death in hospitalized patients. More than 750,000 cases of severe sepsis occur annually in the United States, and the mortality rate is about 30%. As a condition that disproportionately affects the elderly and is related to invasive and immunosuppressive healthcare, increase in the frequency of sepsis is anticipated. The complex pathophysiology of sepsis encompasses the interplay of pro and anti-inflammatory mediators, activation of inflammatory cells, disruption of coagulation, endothelial activation with injury, vasodilatation, and vascular hyporesponsiveness to vasoactive mediators that cause cardiac dysfunction, and cellular dysoxia. Despite the massive research effort over the past two decades to identify innovative therapies, current management of severe sepsis includes eradication of infection through source control and microbial therapy, aggressive and targeted shock resuscitation with fluid administration, correction of anemia, vasopressor support, modest inotropic therapy, and compulsive supportive care to manage organ dysfunction and to avoid complications. Novel therapeutic strategies include use of activated protein C (an endogenous protein that inhibits thrombosis and inflammation while promoting fibrinolysis), small doses of steroids (methylprednisolone) for long period, limiting the tidal volume in Acute Lung Injury / Acute Respiratory Distress Syndrome, an early goal-directed therapy, and a tight control of glycemia with insulin treatment even in non-diabetic patients. It has been demonstrated, in clinical trials, that these strategies can improve survival of septic patients.

Breast cancer is often an estrogen dependent disease and several endocrine treatment options are well established in clinical practice. Although ovarian estrogen synthesis ceases at menopause, estrogens may still be produced by the aromatization of androgens. The “aromatase-pathway” is the major (and probably only) source of estrogens in postmenopausal women. While the antiestrogen tamoxifen has dominated the field of endocrine treatment of breast cancer for decades, novel aromatase inhibitors and inactivators have recently challenged the position of tamoxifen. Non-steroidal aromatase inhibitors like anastrozole and letrozole as well as the aromatase inactivator exemestane have shown to have advantages compared to the traditional drugs like tamoxifen and megestrol acetate. Currently, the aromatase inhibitors and inactivators mentioned above are going to be established as first-line treatment in postmenopausal women with hormonesensitive, metastatic breast cancer. In addition, aromatase inhibitors and inactivators have the potential to be used in the adjuvant setting as well as in breast cancer prevention. This publication gives an overview about the experience made with the most important aromatase inhibitors and inactivators in all stages of hormone-dependent breast cancer focusing on the compounds belonging to the so-called “third generation”: anastrozole, letrozole and exemestane.