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Maintaining adequate energy and mass balance is necessary to the survival and reproduction of any organism. However, nearly all organisms experience periods of catabolism, when insufficient food is being consumed and endogenous stores must be metabolized to provide the difference. The ubiquity of this phenomenon, perhaps an intuitive understanding of the threat that starvation poses to an individual’s survival, and the fact that many organisms seem to "voluntarily" enter long periods of catabolism (fasting) has caused great interest among physiologists to discover and understand the mechanisms organisms have evolved to survive such periods. In wild animals, merely detecting the existence and duration of periods of fasting or starvation can be difficult and much effort has been devoted toward establishing stable isotope analysis (SIA) as a tool for detecting and understanding the duration of catabolic states in wild animals. However, the results of these studies have been mixed. Increasingly, SIA is also being used to investigate the mechanisms organisms use to cope with periods of catabolism: how nutrients are routed, which are recycled, which are used for energy, and how that differs between different organs and organ systems. This chapter reviews the past and current applications of SIA for understanding fasting and starvation in animals and identifies areas where SIA may yet be applied to increase our understanding of fasting and starvation.

Since their discovery in 1895, X-rays have been widely used for imaging humans. Recently, they have also gained an importance in small animal imaging (SAI). Most techniques known from clinical medicine, including single- and dual-energy X-ray imaging, have been successfully ported to SAI and are the subject of this chapter. As trivial as it is, simple X-ray examinations may bring diagnostically valuable information in a variety of applications. Unenhanced radiography reveals skeletal anatomy, contrast-enhanced imaging allows improved visualization of the vasculature and strongly vascularized areas, and dedicated methods such as bone densitometry deliver quantitative information. In analogy to clinical X-ray imaging, we will separately describe standard two-dimensional (2D) projection imaging and the more advanced three-dimensional (3D) computed tomography (CT) imaging techniques. Also in analogy to clinical applications, CT is considered to be of significantly higher importance as it provides more information and possibilities than conventional 2D approaches. It will therefore be covered in much more detail.

Overt hypothyroidism most often causes moderately elevated cholesterol and triglycerides; but, in genetically predisposed individuals, it can also trigger frank hyperlipidemia, e.g. type III hyperlipoproteinemia. In contrast, in subclinical hypothyroidism, T4 lowers low-density lipoprotein cholesterol (LDL-C) by only 3.5–6.0 mg/dl LDL-C for each 1 mU/l thyroid-stimulating hormone (TSH) increase from baseline, a finding barely perceptible in individual patients. In humans, T3 decreases apoB-containing IDL, LDL and chylomicron remnant particles by up-regulation of LDL receptors and despite a concomitant increase in HMGCoA reductase in the liver. In rodents, T3 also up-regulates CYP7A1, ABCG5 and ABCG8 which interact to increase bile acid synthesis and biliary elimination of cholesterol. Thyromimetics, selective for either thyroid hormone-receptor β or liver, lower both LDL-C in humans and HDL-C in mice, thereby promoting reverse cholesterol transport. Interestingly, the thyromimetic T-0681 protects cholesterol-fed rabbits from atherosclerosis. If safety concerns with TRβ-selective thyromimetics can be addressed (i.e. the observed pituitary feed-back inhibition leading to low TSH levels), this class of drugs may complement statins in tackling the burden of atherosclerosis in humans.

All retinoids (vitamin A and its analogs) in the body originate in the diet either as preformed vitamin A, usually from animal food sources, or as provitamin A carotenoids, usually from plant food sources or as supplements or additives to processed foods. Both preformed vitamin A and provitamin A undergo metabolism within the intestine (Blaner and Olson 1994), including a series of metabolic conversions, extracellularly in the lumen of the intestine and intracellularly in the intestinal mucosa, which result in the preponderance of the dietary vitamin A being converted to retinol (vitamin A alcohol). The retinol, along with other dietary lipids in the intestinal mucosa, is packaged as retinyl ester in nascent chylomicrons. The chylomicrons are secreted into the lymphatic system, and approximately 75% of chylomicron retinoid is eventually taken up as part of the chylomicron remnants by the liver, where the majority of the body’s retinoid reserves are stored. Some retinoid is delivered to extrahepatic tissues where this retinoid is either stored as retinyl ester, released back into the circulation for delivery to the liver and other tissues, metabolized to active retinoid forms such as retinoic acid or metabolized to catabolic forms destined for excretion from the body.

Epidemiological evidence indicates that hypercholesterolemia, smoking, diabetes mellitus, and hypertension are major “risk factors” for cardiovascular disease. However, until now the molecular mechanisms of how these and other risks trigger the disease have escaped detection (Scrott et al. 1972; Goldstein and Brown 1977, 1989; Havel and Kane 1989; Steinberg et al. 1989; Hauss 1990; Brown and Goldstein 1986, 1990; Schwartz et al. 1991; Cambien et al. 1992). Furthermore, unlike cancerogenesis with the discovery of oncogenes and tumor suppressor genes, no unifying hypothesis of atherogenesis has emerged (Bishop 1987; Henderson et al. 1991; Weinberg 1985, 1991). This lack of direction is not surprising, because atherosclerosis is a chronic, multicellular, and probably multistep disease that manifests itself in several distinguishable morphological forms as well as in different parts of the arterial tree depending on the risk factor (Ross and Glomset 1974a,b; Ross 1986). Nevertheless, significant progress has recently been made in our understanding of arterial wall biology. These results have shed new light on the potential molecular origins of the disease and in particular on mechanisms that are associated with hypercholesterolemia. In this chapter we will focus on the relation between atherogenesis and hypercholesterolemia.

Apolipoprotein B (apo B)is a large hydrophobic protein which circulates in two distinct forms, each the product of a single apo B gene (Chan, 1992). The small intestine synthesizes and secretes a form of apo B referred to on a centile scale as apo B48, a protein of 264 KDa, which is associated with chylomicron remnants (Powell et al., 1987, Chen et al., 1987). Human liver produces a form of apo B referred to as apo B100. Apo B100, a 550 KDa protein, circulates in association with low density lipoproteins, the major transport vehicle for cholesterol ester in humans (Chan, 1992). This organ- specific production of apo B isophorms has as its basis a unique post- transcriptional modification to the apo B mRNA. Apo B transcription occurs in the liver and small intestine and yields a 24 Kb mRNA species which contains a CAA codon at position 2153, encoding glutamine in apo B 100 and which is specified in the genomic sequence. In the small intestine, this apo B mRNA undergoes a site-specific cytidine deamination which results in the production of a UAA codon and thereby specifies an in-frame translational stop (Powell et al., 1987, Chen et al., 1987). Work conducted over the last few years using isolated intestinal cell extracts and synthetic apo B RNA templates has enabled investigators to determine that apo B mRNA editing is a process mediated by protein(s) and with the characteristics of an enzymatic reaction (Teng and Davidson, 1992).

Treatment of hepatic metastases, predominantely from colorectal carcinoma, is a major problem in Europe and the USA. Each year 140 000 new cases present in the USA (Sugarbaker 1990). Approximately 7000 of these patients require hepatic resection, the only treatment currently providing 25%-50% long-term survival (Sugarbaker 1990; Sugarbaker and Kemeny 1989). Surgical removal with similar favorable results of hepatic metastases from endocrine tumors as well as from renal cell carcinomas, adrenal carcinomas, leiomyosarcomas, melanomas, and pancreatic carcinomas in selected patients has also been reported (Sugarbaker and Kemeny 1989). In addition, hepatocellular carcinoma (HCC), the most common primary liver carcinoma is the leading cause of cancer mortality in Southeast Asia and sub-Saharan Africa, with an extensive number of people affected (Schonland et al. 1985). Similar to hepatic resections for metastases, resection of small (< 5 cm) HCC, when feasible with regard to cirrhosis, has also prolonged the survival of these patients (Ohnishi et al. 1987).

Male rats treated with large amounts of 17-α-ethinyl estradiol develop a pro-found hypolipidemia that affects all of the major lipoprotein classes (Chao et al. 1979). Plasma cholesterol, triglycerides, and the apolipoproteins A-I and E axe lowered to less than 10% of normal. The augmented clearance of low density lipoproteins (LDL) is eliminated in functionally eviscerated animals, indicating that the liver is the site of the increased removal (Chao et all979). This is in line with the observation that isolated perfused rat livers from est- radiol-treated rats catabolize LDL at a much higher rate than controls. Membranes, prepared from livers of estradiol-treated rats, exhibit a 3- to 10-fold increase in saturable binding sites for human LDL (Kovanen et al. 1979). The estradiol-stimulated lipoprotein receptor of the rat recognizes lipoproteins that contain apolipoprotein B-100 (LDL, VLDL, and VLDL remnants) in vivo and in vitro (Windier et al. 1980). The receptor also recognizes synthetic lamellar complexes of lecithin and apolipoprotein E as well as canine high density lipoproteins containing apolipoprotein E (Windier et al. 1980). It is biochemically and immunologically identical with the LDL-receptor of other species (Roach and Noel 1985; Cooper et al. 1987; Jaeckle et al. 1989).

Apolipoprotein (apo) B is required for the normal transport of lipids in the plasma and exists in two forms [6]. ApoB-100 is the major apolipoprotein constituent of very low density (VLDL) and low density lipoproteins (LDL). ApoB-48 is the major form of apoB in chylomicrons. ApoB-100-containing LDL are known to be atherogenic lipoproteins, and have a relatively long plasma residence time. In contrast, apoB-48-containing chylomicron remnants are not clearly atherogenic, and are removed from the plasma very rapidly. ApoB-100 and apoB-48 are encoded by the same gene on chromosome 2 [1,15]. ApoB-100 has a predicted MW of 512 Kd and is a ligand for the LDL receptor [7]. ApoB-48 is approximately half the size of apoB-100 and shares an identical amino terminus with apoB-100, however it lacks the carboxy-terminal domain of apoB-100 [19] which is necessary for LDL receptor binding [7]. The catabolism of apoB-48 is thought to be dependent largely on apoE [13].

The terminal catabolism of most plasma lipoproteins occurs chiefly in the liver. The LDL receptor mediates the removal of apolipoprotein (apo) B-100 containing lipoproteins such as low density lipoproteins (LDL) and their partially catabolized precursors, very low density lipoprotein (VLDL) remnants, from the blood (1,2). Yet, the removal mechanisms for chylomicron remnants and high density lipoproteins (HDL) have still to be solved.

Hydrophobic cholesteryl esters are transported through the aqueous environment of the plasma, encapsulated within a detergent envelope of phospholipid and specific amphipathic proteins (see [1] for review). The latter, the apolipoproteins, not only are responsible for maintaining the structure of the particle but also act as recognition signals for cell membrane receptors which trigger lipoprotein endocytosis [2]. The “classical” lipoprotein receptor, which modulates the metabolism of low-density lipoprotein (LDL), was first described by Goldstein and Brown [3, 4]. It is a trans-membrane glycoprotein with a molecular weight of approximately 96000 daltons which concentrates LDL at specialised areas (coated pits) involved in the endocytic process [5]. Disulphide crosslinks at the extracellular amino-terminal end of the protein are responsible for maintaining the conformation of the ligand binding domain. The intracellular carboxy terminus on the other hand interacts with structural elements underlying the cell membrane which play a key role in generating the coated pit and initiating the uptake process. Natural mutations or laboratory manipulation of the carboxy-terminal amino acid sequence have highlighted the importance of this segment of the molecule in regulating and directing LDL assimilation [6]. The disease familial hypercholesterolaemia (FH) exemplifies the physiological significance of the LDL receptor in modulating corporeal cholesterol homeostasis. Individuals who express this disorder cannot metabolise LDL normally because they lack functional cell membrane receptors for the lipoprotein. Consequently, plasma LDL levels are increased several-fold, exposing the individual to high risk of atherosclerosis [7].

Low density lipoprotein receptor (LDL-receptor) activity in hepatic tissues is difficult to identify and measure accurately by ligand-binding assays that depend upon separation of bound and free ligand by membrane filtration (Schneider et al., 1980) or centrifugation (Basu et al., 1978). With liver membranes, the proportion of non-specific binding relative to specific, saturable binding is high and saturable binding of LDL is less sensitive to EDTA, a potent inhibitor of LDL receptor activity, than in other tissues (Harders-Spengel et al., 1982). The LDL receptor protein in human skin fibroblasts and bovine adrenal cortex has been demonstrated by ligand blotting with LDL (Daniel et al., 1983). The native LDL receptor exhibits Ca++ dependent saturable binding of lipoproteins containing apoB and apoE and these properties are retained in the blotted protein if detergent-solubilised receptor is subjected to SDS-PAGE under non-reducing conditions. We have recently described a new method for the detection of bound lipoproteins based on the high affinity streptavidin-biotin interaction (Wade et al., 1985), and in this paper we report that a protein of MW approx. 130,000–150,000 can be detected in extracts of both adult dog liver and normal rat liver by ligand blotting with biotin-LDL. Binding of the lipoprotein was saturable and EDTA-sensitive, and thus the protein has the properties of the LDL-receptor of human skin fibroblasts or bovine adrenal cortex. Rat chylomicron remnants and rabbit sVLDL, both modified with biotin, were also bound by the LDL-receptor protein in bovine adrenal cortex, dog liver and rat liver.

An abnormal lipoprotein was once defined according to the following criteria: inappropriate lipid composition of density class (by this way the beta-VLDL of type III hyperlipoproteinemia was detected) or unusual protein content of density class (LP (a)). Both criteria led to the detection of lipoprotein X (LP-X).

Lipoprotein receptors, especially those within the liver, play a central role in the regulation of cholesterol metabolism. Specific apolipoproteins (apo-) serve as the ligands for these receptors (for review, see Brown and Goldstein 1983, 1986; Mahley and Innerarity 1983; Mahley et al. 1984).

Virtually 100% of chylomicron remnants are taken up by liver and degraded within lysosomes in hepatic parenchymal cells [1]. Most (> 50–95%) remnants of very low density lipoproteins (VLDL) and most (ca. 50–70%) low density lipoproteins (LDL) are also catabolized by the same metabolic pathway in liver cells [1–3], although remnants of chylomicrons may bind initially to a different receptor than LDL (1 and Mahley, this volume). This enormous traffic of incoming lipoprotein particles is unique to liver and is related to two specialized structures also unique to liver. The first is the sieve plate structure of the sinusoidal lining in which the endothelial cell cytoplasm is perforated by numerous pores averaging about 100–120 nm in diameter, sufficiently large to permit most remnants entry into the space of Disse from plasma and easy access to receptors on hepatocyte microvilli [4].

Epidemiologic and clinical data reveal the suprising fact that more than half of the people in industrialized societies have a level of circulating LDL that puts them at high risk for developing atherosclerosis. The more LDL in the blood, the more rapidly atherosclerosis develops (Goldstein and Brown 1982). However, we do not know yet the ideal level of cholesterol in the human blood. The plasma concentration levels of cholesterol now believed “normal” may well be too high in view of such a prevailing high risk of atherosclerotic lesions in industrialized societies. What determines the blood level of LDL? A specialized protein, called LDL receptor, plays an important role (Goldstein et al. 1983). The number of receptors on the surface of cells varies with the demand for cholesterol of the cells. When the need is low, the cells make fewer receptors and take up LDL at a reduced rate. This will protect cells against excess cholesterol, but at a high price: the reduction in the number of receptors decreases the rate at which LDL is removed from the circulation; the plasma level of LDL rises; and atherosclerosis is accelerated. The liver is a key organ in cholesterol and lipoprotein homeostasis, because of its large size and its high concentration of LDL receptors (Kita et al. 1982). It both synthesizes cholesterol (de novo synthesis) and acquires cholesterol from chylomicron remnants and other lipoproteins. Biliary excretion is the dominant pathway for net removal of cholesterol from the body. The bile acids are largely reabsorbed from the intestine, returned to the bloodstream, taken up by the liver and again secreted into the upper intestine.

Following absorption and esterification by enterocytes in the small intestine, retinol is transported in lymph in chylomicrons [1]. In rats, the retinyl esters are relatively nonexchangeable components of the chylomicrons and their remnants [2]. Accordingly, nearly all the retinyl esters follow the chylomicron remnants when they are taken up by the liver [3]. Several studies have shown that chylomicron remnants are removed from the circulation by hepatic parenchymal cells [4–5], and that these cells express high affinity receptors that recognize the apo E of the chylomicron remnant particles [6–7]. We have studied the hepatic handling of chylomicron remnant retinyl esters. These results indicate that retinol taken up by parenchymal cells as part of chylomicron remnants may have two destinations.

Cholestasis brings about numerous changes in hepatic lipoprotein metabolism. The most pronounced alterations are hypercholesterolemia, hyperlipidemia and appearance of a unique lipoprotein, lipoprotein-X, in plasma. This lipoprotein is characterized by a high content of phospholipids and unesterified cholesterol with a small amount of protein. About 60% of the protein is albumin which is in the core of the vesicle. Apo-C forms the surface apoprotein, whereas apo-B and apo-E are absent. Lipoprotein-X is formed when bile lipids reflux into the plasma [1–4]. Even though LP-X is rich in cholesterol, it is surprising that cholesterol circulating in the form of LP-X does not exert feed-back control through HMG-CoA reducta-se on hepatic cholesterol synthesis during cholestasis. Despite the high cholesterol content of LP-X, its infusion has no significant effect on hepatic cholesterol biosynthesis in normal rats or in rats with a bile fistula [5], in contrast to the inhibitory effect of LDL. As a result of a disturbance in cholesterol homeostasis, hepatic cholesterol synthesis is increased in cholestasis [6]. Its role as a possible causative factor for cholestatic hypercholesterolemia has gained interest in recent years [7].

Our group1 has been investigating the role of the lecithin-cholesterol acyltransferase (LCAT) reaction in plasma lipoprotein metabolism by studying the plasma lipoproteins of patiepts with familial LCAT deficiency.We have performed studies in vivo (1) which suggest that the reaction normally plays a role in the disposal of chylomicron surface lipid. In vitro experiments (2) have shown that the reaction1s effects on plasma lipoproteins are complex, and involve not only the lipids, but also the apolipoproteins and physical properties of the lipoproteins

Triglyceride-rich lipoproteins as very low density lipoproteins and chylomicrons are snythesized by liver and intestine. After their secretion into the bloodstream they are catabolized in two steps. Firstly the triglyceride moiety is hydrolized and tissues are supplied with the released fatty acids. The triglyceride hydrolysis leads to a decrease in particle size and is accompanied by various changes in the chemical composition (Fig. 1).

Continuing advances in our understanding of the metabolism of chylomicrons and very low density lipoproteins (VLDL) in mammals have been critically dependent upon new information about the structure and function of apolipoproteins. This new information has come in substantial measure from the discovery of mutations affecting apolipoproteins or lipoprotein-receptors. Functions can now be ascribed to each of the major groups of apolipoproteins (A, B, C and E) of nascent chylomicrons and VLDL. Here I will review chiefly recent research on the role of apolipoproteins B and E in the metabolism of triglyceride-rich lipoproteins.

A new perspective on lipoprotein metabolism has been gained from the identification and characterisation of Specific high affinity lipoprotein receptors. These are located on cell membranes and serve to facilitate and control the ingress of lipoprotein cholesterol into the cell. They exhibit two important properties. First, they are subject to autoregulation so that when cellular cholesterol requirements rise, the receptors are stimulated to promote lipoprotein assimilation. Conversely, when the cell is replete with the sterol, receptor activity is suppressed. Second, they demonstrate a high degree of ligand specificity which derives from selective interaction between the receptor and apoproteins on the surface of lipoproteins. Two polypeptides, apolipoproteins B and E, have been implicated in this recognition process and two distinct membrane receptor activities have also been identified (1,2,3,4). One seems to be confined to hepatocytes and apparently operates as an efficient mechanism for the cellular assimilation of chylomicron remnants. The other which appears to be identical to the fibroblast receptor of Goldstein and Brown (1) is widely distributed throughout the tissues of the body and plays an important role in the metabolism of low density lipoproteins (LDL).

In vitro and in vivo evidences show that the liver of several animal species and humans catabolizes lipoproteins [1]. In particular two different lipoprotein receptors are expressed by the liver; the E and LDL receptors [2, 3]. While the LDL receptor on the liver is probably identical to the well characterized LDL receptor found in peripheral cells [2, 3] the E receptor seems to be peculiar to the liver and probably mediates the catabolism of chylomicron remnants.

This conference on receptor-mediated uptake in the liver brings together investigators who have studied endocytic mechanisms for a variety of macromolecules. A large emphasis has been placed on the plasma lipoproteins, which in mass terms probably contribute the most to these processes.

Chylomicrons carry lipids of intestinal origin to peripheral tissues and the liver. The lipids are of two sources. First, there are the endogenous lipids of desquamated intestinal epithelial cells and the bile. Secondly, exogenous lipids of dietary origin are absorbed. This latter source is the most important for triglycerides, while much of the cholesterol and phospholipids follow the conservative cycle of endogenous lipids being reabsorbed. As the quantity of triglycerides determines the size of chylomicron particles, postprandially their diameter rises up to 2000 A or more. That is why chylomicrons are postprandially easily recognized in the blood stream, although they are continuously secreted. The particles produced in the fasting state are much smaller with a diameter in the range of 500 A, which is similar to that of very low density lipoproteins (VLDL) of hepatic origin. For this reason these “small chylomicrons” are also referred to as “intestinal VLDL”, which gives rise to confusion as the catabolic pathways of chylomicrons and VLDL differ considerably [1].

Lipoprotein receptors, especially those within the liver, play a central role in the regulation of cholesterol metabolism. Specific apolipoproteins (apo-) serve as the ligands for these receptors (for review, see Mahley and Innerarity, 1983; Mahley et al., 1984; Brown and Goldstein, 1983, 1986).