Physiological roles of CIDEs CIDEA deficient mice exhibit lean phenotype and are obesity resistant 2

Chapter Chapter Introduction to cell death-inducing DFF45like effectors (CIDEs) Chapter 1.1 Apoptosis and DNA fragmentation Cell death is an invariable phenomenon of animal development, and it often continues into adulthood (Raff, 1998) Based on the characteristic ways cells look when they die in different circumstances, it was proposed in 1972 that normal cell death, as well as some pathological death, are, in fact, cellular suicide (Kerr et al., 1972) That is, the cells activate an intracellular death programme and kill themselves in a controlled way - a process now known as programmed cell death, or apoptosis Apoptotic cells shrink and are rapidly phagocytosed by neighbouring cells and macrophage before there is any leakage of their contents This process is so efficient that it is difficult to find an apoptotic cell that is not already been phagocytosed in typical tissue sections (Raff, 1998) The mechanisms of how apoptosis is initiated and executed remained unclear until the molecular identification of the key components of this intracellular suicide program The prototypical apoptotic process can be divided into three phases: an induction phase, the nature of which depends on the specific death-inducing signals, an effector phase, during which the central executioner is activated and the cells become committed to die, and a degradation phase, during which cells acquire the biochemical and morphological features of end-stage apoptosis (Green and Kroemer, 1998) The pattern of this cell suicide programme is evolutionarily conserved from worms to humans (Ellis et al., 1991; Nagata, 1997; Steller, 1995) There are three major components in the program: the Bcl-2 family proteins, the caspases, which belong to a family of cysteine proteases that cleave after aspartic acid residues; and the Apaf1/CED-4 protein that relays the signals integrated by Bcl-2 family proteins to caspases (Adams and Cory, 1998) Death signals originating from the death receptors (such as Chapter TNF and Fas), or the mitochondria trigger the activation of caspase or caspase 9, respectively These activated initiator caspases in turn activate the downstream executioner caspases 3, and The cross-talk between the two pathways is mediated by BID, which upon cleavage by caspase activates the mitochondrial pathway of cell death (Adams and Cory, 1998; Colussi and Kumar, 1999; Slee et al., 1999) A schematic stepwise representation for caspase activation during apoptosis is illustrated in Figure Apoptotic stimuli Death receptors Dexmethasone Mitochondria Pro caspase8 or caspase 10 BID Active caspase or caspase 10 BAX cytochrome c Bcl-2 Apoptosome ( Apaf-1, procaspase 9, cytochrome c) Avtive caspase Pro-effector caspases Active effector caspase 3,6,7 Vital substrates like DFF Apoptosis Figure Apoptotic pathways and caspase activation Death receptors can activate the initiator caspase-8 Activation of caspase-9 by several apoptotic stimuli requires release of cytochrome c into the cytosol from the mitochondria Chapter Upon apoptotic stimulation, initiator caspases such as caspase -8 and -9 are recruited to their respective adaptors, such as FADD and Apaf-1 Through homophilic interactions that result in oligomerization of the initiator caspases, activation is achieved in presumably an autocatalytic fashion Subsequently, activated initiator caspases proteolytically activate downstream effector caspases such as caspase-3 and -7, which in turn carry out the final destruction of the apoptotic cell (Nagata, 1997) The downstream events of apoptosis are characterized by morphological changes that include mitochondrial damage, nuclear membrane breakdown, DNA fragmentation, chromatin condensation, and formation of apoptotic bodies (Takahashi, 1999) DNA fragmentation is often considered a hallmark of apoptosis, a result of the activation of endonucleases that cleave DNA between nucleosomes (Wyllie et al., 1980) 1.2 DNA fragmentation factor (DFF) The factor that is responsible for DNA fragmentation and nuclear condensation in apoptosis has been purified (designated DNA fragmentation factor, or DFF) using an experimental system in which DNA fragmentation was triggered in vitro by activated caspase-3 (Liu et al., 1996; Enari et al., 1998; Liu et al., 1997; Sakahira et al., 1998) DFF is a heterodimeric protein, composed of the 40 kDa caspase activated subunit (DFF40/CAD, CPAN) and its 45 kDa inhibitor subunit (DFF45/ICAD) (Enari et al., 1998; Liu et al., 1997) DFF had been originally purified from a cytoplasmic fraction (Liu et al., 1997), and it was postulated that DFF40 undergoes translocation to the nucleus upon caspase-3 cleavage of DFF45/ICAD Cleavage of DFF45/ICAD by caspase at two different sites releases DFF40/CAD from the complex and triggers DNA fragmentation and chromatin condensation (Enari et al., 1998; Liu et al., 1998) DFF45/ICAD mutants carrying point mutations at either or both caspase-recognition Chapter sites are competitive inhibitors, and cannot be removed upon caspase-3 cleavage to activate DFF40/CAD (Sakahira et al., 1999) Purified DFF40/CAD released from the complex with DFF45/ICAD forms homo-oligomers that are the enzymatically active forms of the nuclease (Liu et al., 1997) DFF40/CAD consists of two domains with distinct functions Its C-terminal part exhibits deoxyribonuclease activity, whereas the N-terminal CIDE-N domain has a regulatory function (Inohara et al., 1998) Similarly, DFF45/ICAD contains an Nterminal CIDE-N domain that is sufficient to inhibit DNA fragmentation (Inohara et al., 1998) The CIDE-N domain represents a 75 amino acid fold consisting of a twisted fivestranded β sheet with two α -helices arranged in an α/β roll (Lugovskoy et al., 1999) The structure of the CIDE-N domain is illustrated in Figure Figure Solution structure of CIDE-N domain (Lugovskoy et al., 1999) DFF45 plays a dual role as both an inhibitor and a chaperon of DFF40 The expression of DFF40 in various systems in the absence of co-expressed DFF45 results in Chapter inactive aggregates This suggests that DFF45 is required for the proper folding of the active DFF40 during its synthesis (Enari et al., 1998; Liu et al., 1997; Liu et al., 1998) The CIDE-N domain of DFF45 is apparently required for its chaperon function, by associating with the CIDE-N domain of DFF40 (Freida L., May 1997; Lugovskoy et al., 1999) A combination of hydrophilic and hydrophobic interactions contributes to the formation of this complex and determines the specificity of the interactions (Zhou et al., 2001) DETD DAVD (caspase-3 cleavage sites) DFF45(nascently translated) DFF40(nascently translated) Aggregated & misfolded DFF40 DFF40/45 complex(folding intermediate ) Cytoplasm DFF40/45 complex(monomeric ) Caspase-3 DFF45 fragments Nucleus Active DFF40 (oligomeric) active nuclease DNA fragmentation Figure Mechanisms of activation of DFF DFF45 can be cleaved by caspase-3 at DETD and DAVD site Both DFF45 and DFF40 are synthesized in the cytoplasm and functions as mutual chaperons The inactive DFF40/45 complex is transfered to nucleus Upon an apoptotic stimulus, DFF45 is cut by activated caspase-3, which releases DFF40 and form active homodimers The activated DFF40 serves as a nuclease to cause DNA fragmentation The molecular structure and mechanism of regulation of DFF are summarized in Figure Human DFF40 is a basic protein with a pI of 9.3 and is composed of 345 Chapter amino acids, while DFF45 is an acidic protein with a pI of 4.5 and composed of 331 amino acids As illustrated in Figure 3, DFF40 and DFF45 are synthesized in the cytoplasm and serve as each other’s folding chaperone The catalytically inactive complex DFF40/DFF45 is then targeted to the nucleus When an apoptotic stimulus triggers the activation of caspase-3, it cleaves DFF45, thereby releasing DFF40 from the complex Released DFF40 forms catalytically active homo-oligomers that degrade chromosomal DNA (Zhou et al., 2001) 1.3 Physiological roles of DFF The physiological significance of DFF in triggering internucleosomal cleavages during apoptosis has been unequivocally demonstrated DFF40 and DFF45 mRNAs and proteins are expressed in most tissues and cell lines Cell lines expressing high levels of DFF40 and DFF45 quickly undergo DNA fragmentation upon an apoptotic stimulus (Mukae et al., 1998) Thymocytes and splenocytes from mice that lack functional DFF45 gene exhibit neither DNA laddering nor chromatin condensation when exposed to apoptotic stimuli (Wu et al., 1999) Although such mice appeared normal, thymocytes from these mice are more resistant to apoptosis than that from wild-type animals (Wu et al., 1999) The degradation of genomic DNA into nucleosomal units is one of the best characterized biochemical hallmarks of apoptosis, and has served as the biochemical basis for commonly used techniques to detect apoptotic cells (e.g., TUNEL (Terminal deoxytransferase-mediated deoxy uridine nick end-labelling) assays) One of the physiological roles of apoptosis is to remove harmful cells (i.e cancer or virally infected) from an organism It was hypothesized that mechanisms of DNA breakdown during apoptosis are developed to prevent transfer of potentially “incorrect DNA” (e.g., Chapter activated oncogenes or viral genes) to another cell or to reduce the possibility of autoimmune responses (Widlak, 2000) It has been shown that cells can die as a result of apoptotic stimuli without internucleosomal DNA cleavage (Cohen et al., 1994; Oberhammer et al., 1993) However, although apoptotic cell death can be disconnected from DNA breakdown, this event is beneficial for the efficient removal of potentially toxic cell debris from the organism 1.4 CIDE family To identify potential DFF45-related genes, Inohara and colleagues searched the expressed sequence tag (EST) database of Genbank for clones with homology to DFF45 and identified a novel family of cell death inducing-DFF45-like effector (CIDE) proteins (Inohara et al., 1998) These CIDEs share an N-terminal domain that is homologous to the CIDE-Ns of DFF40/CAD and DFF45/ICAD (Inohara et al., 1998) CIDE proteins are highly homologous between human and mouse The mammalian family members Cidea, Cideb, and FSP27 share homology with each other both in the CIDE-N and CIDE-C domains as shown in Figure 4A The CIDE-C domain show apoptosis inducing function (Inohara et al., 1998) CIDE-N DFF45 335 32 94 48 108 121 188 219 49 108 125 193 219 56 CIDE-C Cidea Cideb FSP27 116 135 203 239 Figure 4A Schematic structure of mouse Cidea, Cideb, FSP27 and DFF45 Chapter The amino acid sequence alignments of CIDEs are shown below in Figure 4B Figure 4B Sequence alignments of CIDEs family members The accession numbers for hDFF45, hCIDE-A, mCIDE-A, FSP27, hCIDE-B and mCIDE-B are NP_004392, NP_938031, NP_034174, NP_848460, NP_055245, and NP_034024 respectively Red color characters indicate conserved in all molecules and blue color characters indicate conserved in more than two molecules Alignment was performed using Clustalw program Conserved residues are listed in the line labeled as “consensus” hDFF45, hCIDE-A and hCIDE-B are from humans, mCIDE-A, mCIDE-B and FSP27 are from mice Chapter It has been shown that ectopic overexpression of many CIDEs causes apoptosis (Inohara et al., 1998) The C-terminal domain of Cidea is necessary and sufficient for cell killing, whereas its N-terminus is required for DFF45 to inhibit Cidea induced apoptosis (Inohara et al., 1998) Thus it has been postulated that N-terminal domains play a regulatory role in mediating CIDE-induced apoptosis by associating with other CIDE-N functional domain containing proteins (Inohara et al., 1998) Cideb, on the other hand, is localized to mitochondria when overexpressed ectopically and can form homo- or heterodimers with other CIDE family members (Chen et al., 2000) Furthermore, the C-terminal region of Cideb, which shares homology with Cidea and FSP27, is responsible for Cideb induced cell death, mitochondrial localization and dimerization (Chen et al., 2000) Unlike the ubiquitous DFF45 and DFF40, both Cidea and Cideb are expressed in a more restricted manner and show pronounced tissue specificity Expression of human Cidea was detected in heart, skeletal muscle, brain, lymph node, thymus, appendix, bone marrow, placenta, kidney and lung (Inohara et al., 1998) Human Cideb was detected in adult and fetal liver as well as in spleen, peripheral blood lymphocyte and bone marrow (Inohara et al., 1998) Cidea, but not Cideb mRNA, was expressed in 293T embryonic kidney, MCF-7 breast carcinoma and SHEP neuroblastoma cells (Inohara et al., 1998) Although Cidea and Cideb activate apoptosis when ectopically expressed, and appear to function as positive effectors of the apoptotic pathway, it is still not clear which cell death-signaling cascade Cidea, Cideb or FSP27, might be involved Overexpression of Cidea or Cideb alone induced caspase-independent cell death that 10 Chapter person’s obesity may be misleading because obesity can be caused by a variety of genetic and environmental components The mouse syndromes are a dramatic demonstration of the complexity inherent in weight regulation Therapies that target energy intake or expenditure alone may initially produce weight loss, but the existence of a homeostatic feedback loop would be predicted that acts to resist further weight loss and limit efficacy Hence, it seems unlikely that most obesity is caused by the function or malfunction of a single gene 16 Chapter 1.5.3 The liver plays a central role in lipid transport and metabolism Fat absorbed from the diet and lipids synthesized by the liver and adipose tissue must be transported between the various tissues and organs for utilization and storage Since lipids are insoluble in water, the problem arises of how to transport them in an aqueous environment such as blood plasma This is solved by associating nonpolar lipids (triglycerides and cholesteryl esters) with amphipathic lipids (phospholipids and cholesterol) and proteins to make water-miscible lipoproteins (Mayes, 1990) Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties They are chylomicrons derived from intestinal absorption of triglyceride, which transport exogenous (externally supplied, in this case, dietary) triglycerides and cholesterol from the intestines to the tissues; very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low-density lipoproteins (LDL), a group of related particles that transport endogenous (internally produced) triglycerides and cholesterol from the liver to the tissues (the liver synthesizes triglyerides from excess carbohydrates); and high-density lipoproteins (HDL), which transport endogenous cholesterol from the tissues to the liver The protein components of lipoproteins are known as apolipoproteins or just apoproteins At least nine apoproteins are distributed in significant amounts in the different human lipoproteins They are ApoA-I, ApoA-II, ApoB-48, ApoB-100, ApoC-I, ApoC-II, ApoC-III, Apo-D, Apo-E Plasma lipids are composed of four major groups − triglycerides, phospholipids, cholesterol, and cholesteryl esters In addition, a much smaller fraction is in the form of unesterified long-chain fatty acids (LCFA) and free fatty acid (FFA), which are known to be the most metabolically active of the plasma lipids 17 Chapter Lipids are transported in lipoprotein complexes The lipid digestion products absorbed by the intestinal mucosa are converted by these tissues to triglycerides and then packaged into lipoprotein particles called chylomicrons These in turn are released into the bloodstream via the lymph system for delivery to the tissues Similarly triglycerides synthesized by the liver re packaged into very low density lipoproteins (VLDL) and released directly into the blood The triglycerides components of chylomicrons and VLDL are hydrolyzed to FFA and glycerol in the capillaries of adipose tissue and skeleton muscle by lipoprotein lipase (LPL) The resulting FFAs are taken up by these tissues while the glycerol is transported to the liver or kidneys There it is converted to glycolitic intermediate dihydroxyacetone phosphate by the sequential actions of glycerol kinase and glycerol-3-phosphate dehydrogenase Mobilization of triglycerides stored in adipose tissue involves their hydrolysis to glycerol and FFAs by hormone sensitive lipase (HSL) The FFAs are released into the bloodstream, where they bind to albumin, a soluble 66.5-kD monomeric protein that comprises about half of the bloodstream protein Triglyceride is the predominant lipid in chylomicrons and VLDL, whereas cholesterol and phospholipid are the predominant lipids in LDL abd HDL, respectively All plasma lipoproteins are interrelated components of one or more metabolic cycles that together are responsible for the complex process of plasma lipid transport The liver plays a central role in lipid transport and metabolism (Mayes, 1990) The metabolic interaction between liver and other tissues is illustrated in Figure 18 Chapter Exogenous pathway Dietary fat Endogenous pathway Bile acids and cholesterol ApoB-100 Endogenous cholesterol Li ver Intestine LDL LDL receptor LDL receptor Extra hepatic tissue Dietary cholesterol Remnant receptor Chylomicrons ApoE C-II B-48 Remnants ApoE B-48 Capillaries LPL FFA Adipose tissue, muscle VLDL ApoE C-II B-100 IDL HDL ApoE B-100 ApoA-I A-II Plasma LCAT Lecithin Cholesterol Acyl Transferase Capillaries LPL FFA Adipose tissue, muscle Figure Liver plays a central role in lipid transport and metabolism Triglyceride is transported from the intestines in chylomicrons and from the liver in VLDL FFA arises from lipolysis of triglyceride in adipose tissue or as a result of the action of lipoprotein lipase during the uptake of plasma triglyceride into tissues 1.5.4 Adipose tissue is the main storage site for triglyceride in the body In adult mammals, the major bulk of adipose tissue is a loose association of lipidfilled cells called adipocytes, which are held in a framework of collagen fibers In addition to adipocytes, adipose tissue contains stromal-vascular cells including fibroblastic connective tissue cells, leukocytes, macrophages, and pre-adipocytes (not yet filled with lipid), which contribute to the structural integrity Adipose tissue is therefore a specialized connective tissue that functions as the major storage site for fat in the form of triglyceride 19 Chapter Glucose-6-phosphate Glycolysis Acetyl-CoA Citric Acid Cycle CO2 Lipogenesis β-Oxidation Glycerol 3-phosphate Acyl-CoA Esterification ATP CoA Acyl-CoA Synthetase TG HSL FFA FFA (pool 2) (pool 1) Lipolysis Glycerol Adipose tissue LPL Plasma Insulin FFA Glycerol TG(Chylomicrons,VLDL) FFA Glucose Figure Key metabolic cycles in the adipose tissue TG, triglyceride; FFA, free fatty acids; VLDL, very low density lipoprotein; HSL, hormone sensitive lipase; LPL, lipoprotein lipase The triglyceride stores in adipose tissue continuously undergo lipolysis and reesterification (see Figure 7) These two processes are not the forward and reverse phases of the same reaction Rather, they are entirely different pathways involving different reactants and enzymes Many of the nutritional, metabolic and hormonal factors that regulate the metabolism of adipose tissue act either on the process of esterification or lipolysis The resultant of the these two processes determines the amount of the free fatty acid (FFA) in adipose tissue, which in turn is the source and determinant of the level of FFA circulating in the plasma Since the level of plasma FFA has profound 20 Chapter effects on the metabolism of other tissues, particularly liver and muscle, factors regulating the outflow of FFA in adipose tissue exert an influence on metabolism far beyond the tissue itself (Voet, 1995) 1.5.5 Adipose tissue as an endocrine organ Adipose tissue has emerged conceptually as an endocrine organ that is central to the regulation of energy homeostasis It can secrete proteins that exert a pleiotropic effect on metabolism upon diet or environmental stimuli The proteins are involved in glucose and fat metabolism and hence can influence insulin resistance They include leptin, resistin, adiponectin, acylation-stimulating protein, tumour necrosis factor-alpha and interleukin-6 (Simpson, 1996) As mentioned previously leptin is the hormone that reports the status of energy storage in adipose tissues which is encoded by the ob gene, and is an important metabolic regulator Initial work indicates that the leptin gene is expressed only in white adipose tissue (WAT), but subsequent developments have shown it to be expressed at lower levels in other forms of adipose tissue, including brown adipose tissue (BAT) A variety of other tissues (e.g., bone, mammary gland, ovarian follicles, placenta, stomach, muscle and certain fetal organs, such as the heart, bone and mucosa) have now been shown to have leptin gene expression The placenta is also a site of leptin synthesis in humans, rodents and ruminants (Friedman and Halaas, 1998) The structure and function difference of BAT and WAT will be stated in section 1.5.7 21 Chapter 1.5.6 Hormones regulate fat mobilization in adipose tissues Adipocytes express a variaety of receptors that are responsible for hormonal regulation of lipid metabolism under different physiological requirements They include insulin-like growth factor-I (IGF-I), growth hormone (GH), leptin, glucagon, α-adrenergic and β-adrenergic receptors (Simpson, 1996) Among these receptors, the β3-adrenergic receptor is the one that can be activated by catecholamines leading to thermogenesis in BAT (Lowell and Flier, 1997) A principle action of insulin in adipose tissue is to inhibit the activity of the hormone-sensitive lipase (HSL), reducing the release of both FFA and glycerol Simultaneously, insulin can enhance the uptake of glucose into adipose tissues, promoting lipogenesis Adipose tissue is thus a major site of insulin action in vivo (Voet, 1995) Other hormones accelerate the release of FFA from adipose tissue and raise plasma FFA concentration by increasing the lipolysis of triglyceride stores (Voet, 1995) These hormones include epinephrine, norepinephrine, glucagon, adrenocorticotropic hormone, thyroid stimulating hormone, growth hormone and vasopressin The hormones that act rapidly in promoting lipolysis, i.e., catecholamines, so by stimulating the activity of adenylate cyclase, the enzyme that converts ATP to cAMP (Voet, 1995) CAMP, by stimulating cAMP-dependent protein kinase (PKA), phosphorylates HSL The activated HSL stimulates lipolysis in adipose tissue, raising blood FFA levels and ultimately activating the β-oxidation pathway in other tissues such as liver, muscle and BAT (Voet, 1995) In liver, this process leads to the production of ketone bodies that are secreted into bloodstream for use as an alternative fuel to glucose by peripheral tissues PKA, acting in concert with AMP-dependent 22 Chapter protein kinase (AMPK), also causes the inactivation of acetyl-CoA carboxylase (ACC), one of the rate determining enzymes of fatty acid synthesis (Voet, 1995) Thus cAMP-dependent phosphorylation simultaneously stimulates FFA oxidation and inhibits fatty acid synthesis The AMPK is the downstream component of a protein kinase cascade that is activated by a rise in the AMP: ATP ratio AMPK is switched on by cellular stresses that either interfere with ATP production (e.g hypoxia, glucose deprivation, or ischemia), or by stresses that increase ATP consumption (e.g muscle contraction) Hormones that act via Gq-coupled receptors, and by leptin and adiponectin, via mechanisms that remain unclear, also activate AMPK It was reported that leptin stimulates FFA oxidation by activating AMPK (Minokoshi et al., 2002) A schema on the leptin and adrenergic signaling pathways in brown adipocytes is shown in Figure 23 Chapter Cold is sensed by the brain Leptin Sympathetic nerves are activated α-adrenergic receptor Ob-Rb Norepinephrine β-adrenegic receptor Adenyl cyclase AMPK AMPKK Activation AMPK-P ACC cAMP ATP HSL ACC-P R2C2 (inactive PKA) 2C (active PKA) R2(cAMP)4 Triglyceride HSL-P (active) FFA Malonyl-CoA Acetyl-CoA Fatty acyl-CoA CPT1 H+ Brown adipocyte H+ Respiratory chain H+ ATP Synthase ADP ATP H+ H+ H+ Fatty acyl-CoA Citric Acid Cycle β-Oxidation Acetyl-CoA Heat UCP1 H+ Figure the mechanism of hormonally induced uncoupling of oxidative phosphorylation in brown fat mitochondria The sympathetic nervous system (SNS), through liberation of norepinephrine in adipose tissue, plays a central role in the mobilization of FFA by exerting tonic influences, even in the absence of augmented nervous activity (Lowell and Flier, 1997) 24 Chapter It was established that β-adrenergic stimulation of the tissue leads to rapid hydrolysis of the multi triglyceride droplets within the brown adipocytes and that the abundant mitochondria are able to oxidize the resulting fatty acids at an enormous rate to account for the heat production 1.5.7 Brown adipose tissue promotes thermogenesis Adipose tissue is found in mammals in two different forms: white adipose tissue (WAT) and brown adipose tissue (BAT) As shown in Figure 9, they have dramatically different morphological appearances WAT consists of unilocular cells containing a single large lipid droplet that pushes the cell nucleus against the plasma membrane, giving the cell a signet-ring shape (Fig lower picture) Mitochondria are found predominantly in the thicker portion of the cytoplasmic rim near the nucleus The large lipid droplets not appear to contain any intracellular organelles Multilocular cells, typically seen in BAT, contain many smaller lipid droplets (Fig upper picture) The brown color of this tissue is derived from the rich vascularization, and densely packed mitochondria that have extensively developed cristae These mitochondria vary in size and may be round, oval, or filamentous in shape WAT is not as richly vascularized as BAT, but each adipocyte in WAT is in contact with at least one capillary This blood supply provides sufficient support for active metabolism, which occurs in the thin rim of cytoplasm surrounding the lipid droplet Blood flow to adipose tissue varies depending upon body weight and nutritional state, with blood flow increasing during fasting, presumably to transport the products of lipolysis (FFA and glycerol) to the other tissues 25 Chapter Brown Adipose tissue(BAT) Energy expenditure Adaptive thermogenesis antiobesity White Adipose tissue(WAT) Energy storage Obesity Figure Paraffin sections of adult mice BAT (upper) and WAT (lower) stained with haematoxylin & eosin BAT is found in various body locations, depending upon the species and age of the animal In mice, BAT is found primarily in the interscapular region on the back and the axillae around neck, with minor amounts found near the thymus and in the dorsal midline region of the thorax and abdomen Newborn mammals that lack fur, such as humans, as well as hibernating mammals, contain BAT in their neck and upper back that functions in regulating body temperature via non-shivering thermogenesis (Himms- 26 Chapter Hagen, 1990) Adult humans not contain a significant amount of BAT, but other mammals, such as mice contain appreciable amounts of BAT throughout their life WAT and BAT are distinct from a morphological perspective, as described above BAT is highly vascular and intensely innervated by sympathetic nerves However, the most unique feature of BAT is that all differentiated brown adipocytes express an uncoupling protein UCP1, a protein dimer of 32 kDa monomers which is apparently absent in the mitochondria of other tissues (Gura, 1998; Klingenberg and Echtay, 2001) UCP1 spans the inner mitochondrial membrane and functions as an H+ channel to dissipate the proton electrochemical potential gradient as heat (depicted in Fig 8) The flow of protons through this channel is inhibited by physiological concentrations of purine nucleotides (ADP, ATP, GDP, GTP), but FFA can overcome this inhibition The concentration of FFA in BAT is controlled by the hormone norepinephrine liberated from sympathetic nerve endings, with cAMP acting as a second messenger (Fig 8) Under norepinephrine stimulation, the adenylate cyclase synthesizes cAMP, which in turn activates HSL via the activation of kinases The activated lipase hydrolyzes triglycerides to yield FFA that open the proton channel 1.5.8 Control of energy expenditure-adaptive thermogenesis by BAT Adaptive thermogenesis as one component of energy expenditure is of particular interest in the control of obesity In yeast, the uncoupling activity of UCP1 and other uncoupling proteins had been studied extensively (Bouillaud et al., 1994) (Fleury et al., 1997;Rial et al., 1999;Klingenberg and Echtay, 2001) The role of UCP1 as an uncoupler and role of the sympathetic nervous system in activating BATmediated thermogenesis via β-adrenergic receptors and cAMP are well established 27 Chapter Whether BAT or UCP1 has any role in regulating body weight has been further investigated by the several genetic models described below Lowell et al constructed transgenic mice with a DNA plasmid in which the mouse UCP1 promoter region drove expression of the diphtheria toxin A chain (UCPDTA) (Lowell et al., 1993) The expression of the toxin gene selectively in brown adipocytes would destroy these cells The UCP-DTA mice are hyperphagic, obese and have a phenotype characteristic of type II diabetes UCP-DTA mice exhibit cold sensitivity and also have a reduced core body temperature and metabolic rate In addition, UCP-DTA mice exhibit increased plasma leptin levels and resistance to its actions in a way that is similar to the db/db mice (Lowell et al., 1993) Another transgenic line of mice expressing UCP1 in both brown and white adipose tissue was driven by the aP2 promoter (aP2-UCP) (Kopecky et al., 1996a) More than 95% of brown adipocytes are lost in these mice, possibly because of the toxic effects of UCP1 when present in the mitochondria at excessive levels The mice are neither hyperphagic nor obese They exhibit resistance to diet-induced obesity and diabetes, which could be due to increased energy expenditure arising from ectopic expression of UCP1 in WAT (Kopecky et al., 1996b) Targeted inactivation of the UCP1 gene by homologous recombination was also performed in mice to test the effect of thermogenesis on body weight Mice lacking UCP1 showed rapid decrease of core body temperature during cold exposure but no weight gain (Enerback et al., 1997; Klaus et al., 1998) Increased adiposity was apparent in interscapular BAT of UCP1-deficient mice, a finding consistent with the inability of the tissue to utilize its lipid deposits for thermogenesis The BAT of UCP1deficient mice also expresses a five fold elevation of UCP2 mRNA levels, which may be a compensatory mechanism for the regulation of adiposity (Enerback et al., 1997) 28 Chapter Nearly all-experimental rodent models of obesity are accompanied by diminished or defective BAT function (Himms-Hagen, 1990) Disrupting BAT function by denervation or excision of interscapular BAT led to increased mouse body weight (Dulloo and Miller, 1984) Deletion of PKA (R2C2 as shown in Fig 8) regulatory subunit II β (PKA RII β), which is abundantly expressed in BAT, WAT and brain, resulted in elevated UCP1 in BAT, likely a consequence of a compensatory response in the targeted mice (Cummings et al., 1996) The PKA RII α regulatory subunit, an isoform with higher avidity for cAMP, is synthesized at elevated levels in these mice and causes an increase in UCP1 levels These mice showed higher basal energy expenditure, resulting in elevated body temperature and reduced adiposity (Cummings et al., 1996) β3-Adrenergic Receptors (β3-ARs) are abundant in white and brown adipose tissues of rodents β3-ARs are the receptors of norepinephrine on BAT to stimulate thermogenesis Mice that were deficient in β3-AR show slightly increased fat stores (female more than males) Acute treatment of wild-type mice with CL316, 243, a β3selective agonist, increased serum levels of FFA and insulin, increased energy expenditure, and reduced food intake despite reductions in serum leptin concentration Mice lacking all three β-adrenergic receptors have a reduced metabolic rate due to the lack of diet-induced thermogenesis and developed obesity when fed with a high fat diet (Bachman et al., 2002) Generally speaking, BAT is a remarkable tissue that plays a critical role in regulating body fat stores in rodents There are strong correlations between BAT and obesity However this effect cannot be simply explained by UCP1’s thermogenic 29 Chapter function alone There may be additional mechanisms in BAT that control energy expenditure and metabolic rates in addition to UCP1 1.6 Rationale and aim of research Gene targeting by homologous recombination has been widely used, particularly on mouse embyonic stem (ES) cells, to make a variety of targeted mutations so that the phenotypic consequences of specific genetic modifications can be assessed at the organism level Despite the fact that ectopic overexpression of Cidea and Cideb could individually induce apoptosis in cells, many questions pertaining to their exact contributions to apoptosis in vivo have yet to be answered Cidea was found to be highly expressed in BAT This observation prompted us to generate Cidea null mice to analyse its physiological roles in vivo More interestingly, its homologue Cideb was expressed mainly in kidney and liver, which plays pivotal role in lipid metabolism, while FSP27 is found in both WAT and BAT The expression pattern of CIDE family proteins gives us a hint that their physiological role might be more related to regulation of lipid metabolism rather than apoptosis The aim of this project is to resolve the physiological roles of CIDEs by creating and analysing animal models with targeted gene disruption of Cidea or Cideb (knock out mice) It was postulated that the analysis of these Cidea or Cideb deficient mutants should help to clarify the physiological functions of Cidea and Cideb Here, the procedure for generating of targeted-disruption mice for both Cidea and Cideb will is reported The biochemical and physiological analysis of Cidea knock out mice (Cidea-/- or Cidea null) are thus the foci of this thesis 30 ... targeted-disruption mice for both Cidea and Cideb will is reported The biochemical and physiological analysis of Cidea knock out mice (Cidea- /- or Cidea null) are thus the foci of this thesis 30... 56 CIDE-C Cidea Cideb FSP27 116 135 20 3 23 9 Figure 4A Schematic structure of mouse Cidea, Cideb, FSP27 and DFF45 Chapter The amino acid sequence alignments of CIDEs are shown below in Figure... obese and have a phenotype characteristic of type II diabetes UCP-DTA mice exhibit cold sensitivity and also have a reduced core body temperature and metabolic rate In addition, UCP-DTA mice exhibit