Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Immunology, Endocrine and Metabolic Agents) - Volume 8, Issue 4, 2008

The National Institutes of Health (U.S.A.) identifies several major causes/risk factors as essential contributors to the global epidemic of obesity. These include dysregulated energy balance (more energy intake than energy output), genes and family history, environment, health conditions, medicines, emotional factors, aging, and lack of sleep. [1]. One risk factor that can be controlled is energy balance. This requires leveling of the “teeter-totter” of fuel supply and fuel expenditure. Physical inactivity, as a result of a sedentary lifestyle or the consequence of another illness, is a major contributor to the imbalance, resulting in a shift toward overweight and obesity. Additional contributors are altered utilization of fuel supplies as occurs in metabolic disorders such as Diabetes. Even under those circumstances, weight gain requires fuel intake that exceeds its eventual utilization. Here is where the study of Appetite takes center stage in the battle against the obesity. The promise of pharmacologic manipulation of appetite in healthy individuals and, importantly, in patients with metabolic disorders, has driven the study of the central nervous system regulation of feeding. Not surprisingly, factors discovered to influence hunger/satiety by central nervous systems actions also play important roles in the peripheral utilization of dietary fuel supplies. In this Special Issue, an attempt was made not to comprehensively detail the literature on Appetite, but instead to provide several key examples of how the studies of fuel intake and fuel utilization have converged. This is in large part due to the realization that peptide hormones of both peripheral and central nervous system (CNS) origin significantly affect both sides of the “teeter-totter.” The Issue begins with a review of how factors that affect Appetite exert their actions within the CNS. In that review, Ferguson and Sharkey (Kingston and Calgary, Canada) identify the five potential mechanisms by which satiety factors can reach those centers, highlighting the importance of signals arriving via the afferent limb of the Vagus nerve and blood brain barrier free sites in CNS, known as sensory circumventricular organs (CVOs). Gibson and Korbonits (London, United Kingdom) then describe the multiple neuropeptides reported to affect food intake and appetite regulation and detail the intriguing concept of competing and potentially opposing hormones derived from the same gene product, ghrelin and obestatin. Here the concept of post translational modification of products of the same gene identifies a novel approach to the understanding of appetite regulation and possible modification. Can those post translational modifications be therapeutic targets, in this case to octanoylation of ghrelin by the recently described enzyme, ghrelin 0-acyltransferase (GOAT)? Already pharmacologic tools capable of modifying the activity of this enzyme are available for testing in experimental models. The importance of aminergic signaling in CNS to appetite regulation has been recognized for decades and selective agonists and antagonists have been developed for the study of satiety. This has led to novel classes of drugs including selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors (SSRIs) and 5-HT/noradrenalin reuptake inhibitors (SNRIs) that hold promise as appetite suppressants. In the review by Nonogaki (Sendai, Japan), the promise as well as the complications of clinical use of these potent compounds is detailed, providing insight into the difficulty of targeting CNS appetite regulation in isolation, without secondary consequences. The Special Issue concludes with two reviews that detail the convergence of appetite regulation and glucose utilization. Bello and Moran (Baltimore, USA) detail the actions of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion and participate in the regulation of gastric emptying. Acting as the “ileal brake,” GLP-1 contributes to the CNS regulation of food intake by providing via the Vagus nerve, the afferent signal of satiety (i.e. fullness). In addition this hormone is produced in brain, where it interacts with known feeding pathways to suppress appetite. Potential therapeutics have been developed that either prolong the half life of GLP-1 by inactivating the enzymes that process the peptides, or by developing enzyme resistant peptide analogs. Some of these have been approved by the FDA for clinical use as adjunctive therapy in non-insulin dependent diabetes mellitus (NIDDM). Development of these analogs relied upon the knowledge of the mechanisms, detailed in the Ferguson and Sharkey review, of how agents administered peripherally might access CNS satiety centers. The Special Issue concludes with a comprehensive description by Parkes and colleagues (San Diego, USA) of how pharmaceuticals are developed to treat glucose metabolism and body weight regulation. The authors chronicle the development of amylin analogs as therapies for diabetes and weight management. This review highlights the importance of combinatorial chemistry and the availability of appropriate animal models to the development of effective therapeutic candidates.

In this review we will consider how central structures involved in the regulation of energy balance gather information from the variety of different peripherally derived signaling molecules that we now believe provide an integrated perspective of energy status of the organism. The existence of the blood brain barrier means that the CNS is theoretically unable to directly monitor many of these circulating signals such as adiponectin, amylin, cholecystokinin (CCK), glucose, ghrelin, leptin, and peptide YY (PYY) which do not freely diffuse across this barrier. Studies have identified five primary mechanisms through which these circulating signals may transmit the afferent information they carry from the periphery to the CNS which we describe below. We will both discuss mechanisms and potential contributions of vagal afferent signaling, peptide transporters, vascular endothelial cell signaling, the arcuate nucleus of the hypothalamus, and brain structures which lack the blood brain barrier known as the sensory circumventricular organs (CVOs), in providing sensory information to the integrative centers of the hypothalamus and medulla which play such essential roles in the regulation of energy balance.

Obesity has increasingly become a major medical and public health burden in both developed and developing countries. Numerous hormones are involved in controlling appetite and food intake via the neuro-humoral gut-brain axis and several peptides that are synthesized and secreted from the gut have been recently investigated in order to determine their effects on food intake and satiety. Ghrelin is an appetite-stimulating peptide that rises in the fasted state causing increases in food intake. Obestatin is a newly discovered hormone encoded by the same gene as ghrelin, and its putative role in the regulation of appetite and metabolism is unclear. A number of studies using animal models have shown that obestatin opposes the effects of ghrelin on food intake whilst others found that obestatin has little if any affect on gastrointestinal motility, or on food intake. There are also controversies to the receptor mediating obestatin effects with the previously orphan G protein coupled receptor GPR39 suggested to be the main candidate. Investigating the molecular mechanism underlying obestatin and other gut hormones involved in appetite regulation may prove to be useful in finding new strategies to treat obesity.

Brain serotonin (5-hydroxytryptamine; 5-HT) systems have been implicated in the leptin-independent neural regulation of appetite, as indicated by the anorectic effects of drugs that directly or indirectly stimulate postsynaptic 5-HT receptors. The identification of 5-HT subtype receptor functions in the regulation of feeding behavior and energy balance using genetic models has shed new light on the poorly understood area of leptin-independent appetite regulation. With the voluntary withdrawal of d-fenfluramine, which inhibits serotonin (5-HT) uptake and induces 5-HT release, due to primary pulmonary hypertension, research attention has been focused on the downstream pathways of central 5-HT subtype receptors, especially 5-HT2C receptors (5-HT2CRs), in the regulation of satiety. Central 5-HT systems which operate via 5- HT2CRs have a complex neural network, with certain neurons in the hypothalamus harboring appetite regulatory neuropeptides such as proopiomelanocortin (POMC) and corticosterone releasing hormone (CRH). The 5-HT drugs, which enhance 5-HT release via 5-HT2CR in the hypothalamus, induce the activation of hypothalamus-pituitary adrenal (HPA) axis as well as satiety. On the other hand, the selective 5-HT reuptake inhibitors (SSRIs) and 5-HT and noradrenalin reuptake inhibitors (SNRIs) induce satiety without stimulating the HPA axis. 5-HT2CR and 5-HT1BR substantially contribute to the anorexic effects of fenfluramine, whereas inactivation of 5-HT2CR enhances the effects of SSRIs so as to increase extracellular 5-HT in the brain and induce the appetite suppressing effects via 5-HT1BR. Moreover, SNRIs induce satiety independently of 5-HT2CRs and 5-HT1BRs. alpha-1 Adrenoceptors likely contribute to the appetite suppressing effects of SNRIs. Thus, the complex 5-HT network systems orchestrate leptin-independent central appetite regulation.

Glucagon-like peptide - 1 (GLP-1, 7-36 amide) is an intestinal hormone that is released in response to the luminal presences of nutrients. GLP-1 and related mimetic drugs are potent stimulators of pancreatic insulin, a response that is beneficial for the management of non-insulin dependent diabetes mellitus. Additional investigations have revealed that GLP-1 is an endocrine mediator of the ileal brake and has direct effects on the neural controls of food intake. The overall mechanism of GLP-1 induced food intake suppression is mediated by distinct, but overlapping, peripheral and central systems. GLP-1 is also expressed in the brain and roles for central GLP-1 in feeding control have been proposed. This review will focus on meal-related GLP-1 release, receptor function, synthetic agonists, and peripheral and central mechanism involved in GLP-1 induced inhibition of food intake.

We briefly summarize evidence from non-clinical and clinical studies that amylin agonism has a physiological role in glucose and body weight regulation. Next, the amylin analog pramlintide is highlighted as part of an integrated neurohormonal therapeutic approach in both diabetes and weight management. Finally, attributes of, and analoging strategies to, the amylin molecule are discussed with the goal of improving targeted properties leading to the development of an optimized therapeutic candidate.

Signals triggered through the receptors on the plasma membrane of innate and adaptive immune cells are regulated in space and time (spatial-temporal). While the basic understanding of the temporal events of the signaling cascade initiated at the plasma membrane has remained a longstanding focus, the insights into the spatial distribution of signaling proteins and their reorganization during signaling process is under intense investigation. It has been proposed that membrane rafts on the plasma membrane provide a platform for the organization of signaling molecules during initiation and/or regulation of membrane signaling in a variety of cell types. While the merit of methods to investigate these nanometer size domains are being debated, the details of their role in signal transduction remain a hot topic of investigation. In a Keystone Symposium on “Lipid Rafts and Cell Function” in 2006 membrane rafts were defined as small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions. Small dynamic and compositionally heterogeneous nature of membrane rafts has been proposed to be central to their functional role. A number of signaling proteins are either housed in membrane rafts or traffic through these cholesterol-rich nano-domains during cell signaling. It has been suggested that small and dynamic nature of each membrane raft on an un-stimulated plasma membrane may be key to its existence as “incomplete signaling unit” and therefore contributing to the quiescent state of the cell. Coalescence of membrane rafts on the plasma membrane during cell stimulation allow congregation of signaling proteins in the rafts, thereby promoting their molecular interactions and generation of signals that cascade to the cell interior. Over the past decade it has become increasingly clear that signaling through pattern recognition receptors (PRR) in innate immune cells (e.g., dendritic cells (DC) and macrophages) and multi-chain antigen receptor in cells of adaptive immunity (e.g., B and T cells) either get initiated in membrane rafts or propagated through these nano-domains. While the details related to the involvement of membrane rafts are currently being worked-out, this new paradigm in cell signaling has direct implications in initiation/regulation of immune response during normal and abnormal immune responses. This thematic issue on “Membrane Rafts and Signaling” reviews some important aspects of raft biology and provides an additional possibility of rational drug design to interrupt or modulate signals in immune cells. Kazuhisa Iwabuchi and colleagues from Juntendo University, Graduate School of Medicine in Chiba, focus their review on Lactosylceramide (LacCer, CDw17), an innate immune receptor expressed on neutrophils, that bind a variety of microorganisms, including Bordetella pertussis, Helicobacter pylori, and Candida albicans. This review also provides insights into co-localization of LacCer and a Src kinase, lyn, in membrane rafts and its role in superoxide generation, chemotaxis, and nonopsonic phagocytosis. Katharina Gaus and her colleagues from University of New South Wales in Sydney discuss the role of membrane rafts in phagocytosis mediated through Fc receptors in macrophages, another component cell of innate immunity. Helper CD4+ T cells orchestrate adaptive immune response by providing requisite help to other immune cells. CD4+ T cells are capable of rendering this help only after sensing a pathogen or allergen presented to them by antigen presenting cells. The two interacting cells at their contact site generate immunological synapse (IS). Immune receptors that drive T cell adhesion and activation show remarkable organization at the IS. Recent findings provide evidence for condensation of T cell membrane at the contact site with antigen presenting cells during these cellular interactions. Katharina Gaus and her colleagues highlight a connection between membrane rafts and process of plasma membrane condensation at the IS. The process of membrane condensation in T cells is dependent on polymerization of a cytoskeletal protein, actin. While inhibitors of cholesterol have been widely used to illustrate the importance of these membrane domains in variety of physiological processes, this review discusses the use of oxysterol 7-keto cholesterol (7-KC). This cholesterol molecule offers a perpendicular protruding ketone group in position 7 and its incorporation into the membrane prevents the formation of membrane rafts by decreasing the lipid order and increasing the bilayer polarity. 7-KC appears to be a promising agent for disrupting membrane proximal signaling events that either get generated in the membrane rafts or relayed through these nano-size domains.

Over the last 30 years, many studies have indicated that glycosphingolipids expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, glycosphingolipids form membrane microdomains (lipid rafts) with cholesterol, sphingomyelin glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules. Among the glycosphingolipids, lactosylceramide (LacCer, CDw17) can bind to various microorganisms, including Bordetella pertussis, Helicobacter pylori, and Candida albicans. LacCer is highly expressed on the plasma membranes of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. Neutrophils express several types of PRR, among which CD11b/CD18-integrin (αM/β2-integrin, CR3, Mac-1) plays a central role in the immunological functions of phagocytes. CD11b/CD18 mediates neutrophil phagocytosis of C3bi-opsonized microorganisms via the binding of CD11b/CD18 with C3bi, while CD11b/CD18 is able to bind to PAMPs as a PRR, and mediates phagocytosis of non-opsonized microorganisms. However, CD11b/CD18 has no signaling motif in the cytoplasm to mediate the outside-in signaling by itself. Therefore, other PRRs or signaling molecules are needed to mediate CD11b/CD18- dependent functions. In this review, we introduce the organization and functions of LacCer-enriched membrane microdomains and the roles of these microdomains in CD11b/CD18-dependent neutrophil phagocytosis of non-opsonized microorganisms.

Engagement of surface receptors on immune cells triggers intracellular signaling cascades and downstream activation responses. To function in immune responses, activation of macrophages and T-cells must be finely controlled and regulated. Curiously, different extracellular signals often trigger the same signaling pathways with common elements yet result in vastly different outcomes. It has thus been hypothesized that it is the organization of signal transduction processes in space and time that ultimately determines cell responses. The idea that membrane domains on the cell surface could influence the behavior of signaling proteins, and thus act as ‘signaling organizers’ has merged the fields of membrane biology and signal transduction. It is now known that lipids and proteins are not homogeneously distributed in cell membranes giving rise to specialized membrane domains or lipid rafts. Different concepts of membrane organization put forward lipid-lipid, lipid-protein or membrane-skeleton interactions as the underlying principle for domain formation. This review discusses experimental approaches, and their limitations, that have been used to test these concepts. We further review the role of lipid domains during phagocytosis by macrophages and T-cell activation. Although there are substantial differences between these two cell types, there is strong evidence that receptor clustering induces the coalescence of lipid domains that play an integrated role in signal transduction.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a prevalent phosphoinositide in the inner leaflet of the plasma membrane. PIP2 associates with an ever-growing list of proteins, and participates in a variety of cellular processes. PIP2 signaling to the actin cytoskeleton transduces specific signals necessary for changes in morphology, motility, endocytosis, exocytosis, phagocytosis, and cell activation. The mechanism(s) by which PIP2 signaling pathways are specific is a topic of intense investigation. One working model is the compartmentalization of PIP2-mediated signaling by concentrating PIP2 in cholesterol-dependent membrane rafts, therefore providing spatial and temporal regulation. Here we discuss properties of PIP2 signaling to the actin cytoskeleton in immune cell functioning, the association of PIP2 cellular pools with membrane rafts, and recent work investigating models for compartmentalization of PIP2-mediated signaling in membrane rafts to the actin cytoskeleton.

The presence of microdomains (i.e., lipid rafts) in the plasma membrane and their proposed role(s) in the signal transduction by cell surface receptors, particularly in immune cells, has elicited broad interest yet much debate. Here, we aim to review some observations made by our and other groups over past years on the structural and functional properties of membrane microdomains, focusing mainly on T cells and more particularly on activation of signaling cascades triggered by T-cell receptor (TCR) and Fas/CD95, respectively. We will focus on the progress made at both the experimental and conceptual levels. We will then discuss our recent studies on the identification and characterization of nano-sized membrane domains in the plasma membrane of living cells using an original fluorescent correlation spectroscopy (FCS) approach.

In normal and tumoral B-lymphocytes, proximal signaling requires assemblies of proteins to generate prosurvival and anti-apoptotic information. B-cell receptor (BCR) engagement by antigen results in the formation of raftbased microclusters that incorporate the Lyn kinase. Further involvement of the Syk kinase and the B-cell linker (BLNK) adaptor depend on the kinase activity of Lyn to phosphorylate specific motifs in the cytoplasmic portion of the BCR complex, and to maintain this complex in its “open” conformation. Signaling proceeds by way of phospholipase-Cγ activation, protein kinase Cβ activation and phosphorylation of the “caspase-recruitement domain (CARD)-membraneassociated guanylate kinase (MAGUK) protein-1” (CARMA1) protein. Oligomerization of phosphorylated CARMA1 proteins generates a CARMA1 signalosome, which is anchored in rafts and permits phosphorylation of the inhibitor of nuclear factor kappa-B (NF-κB), its degradation in the proteasome and activation of NF-κB. Constitutively active signalosomes have been identified in B-lymphoma cell membranes that either lock the Lyn kinase in a permanently active configuration by association with the Csk-binding protein (Cbp) Cbp/phosphoprotein associated with glycosphingolipid-rich domains (PAG) adaptor, or maintain the CD40 receptor permanently active by engagement with its ligand CD154. The constitutively active Lyn is directly connected with pro-survival and anti-apoptotic pathways, as Lyn inhibition results in lymphoma cell death and the CD40-CD40 ligand signalosome sends co-stimulatory signals via the NF-κB pathway. The constitutively active signalosomes exploit raft microdomains to assemble signaling proteins that generate inappropriately durable signals. Such signalosomes reflect the dependence of lymphoma cells on a narrow spectrum of signaling proteins for the generation of survival signals, and could be exploited therapeutically.

Membrane rafts, due to the presence of several immunoreceptors, signal-transducing kinases and lipids such as ceramides which can act as second messengers, play a crucial role in the cell signaling network which fine-tunes various biological effects. The ability of membrane rafts to segregate receptors provides a mechanism for compartmentalization of signaling molecules in plasma membrane by concentrating some components in membrane rafts and excluding others. Based on these observations, the concept of raft-based therapeutics has recently emerged. Raft-targeting molecules can modulate the lipid-protein rheostat of membrane rafts to treat various diseases, such as cancer, neurological disorders or infectious diseases. In this review, we focus on membrane rafts as “discrete” organizing elements of T lymphocytes' plasma membrane and how to reconcile the dynamic nature of membrane rafts with the formation of the immunological synapse. We describe CD4-specific antibodies as prototypical modulators for the disruption of the lipid-protein rheostat in membrane rafts and extend the concept of raft-based therapeutics to other antibodies, sterol- and sphingolipid-modulating drugs, glycerophospholipid analogs, fatty acid modulators, and peptide-derived molecules. This review highlights a novel mode of action of drugs through dietary or therapeutic interventions that target membrane rafts.