By direct biochemical methods, we demonstrate that the process of internalization of receptors for epidermal growth factor (EGF) occurs even without EGF stimulation and is not prevented by the lysomotopic agents methylamine or chloroquine. These agents inhibit the degradation of 12I4abeled EGF, thus preventing the rapid dissociation of EGF from cells. Furthermore, ls25IabeledEGF incubated with cells in the presence of methylamine becomes increasingly insensitive to trypsin with time, suggesting that the EGF receptor internalization is not preventec by alkylamines, but that there is an intracellular accumulation of ligand-receptor complex due to the loss of normal modes of ligand-induced receptor processing. Lysis of cells treated with methylamine results in recovery of 25I-labeled EGF binding. Fractionation of these lysates on sucrose density gradients demonstrates that EGF receptors are localized within membrane fractions having higher densities than fractions from lysates of untreated cells. Cultured human and murine fibroblasts can be stimulated to synthesize DNA by epidermal growth factor (EGF), a potent mitogen (1, 2). The mitogenic response depends primarily upon the initial interaction of the hormone with specific cell surface receptors (1, 3, 4) but displays a long characteristic lag period (16)(17)(18)(19)(20) hr) before the effect can be detected (4, 5). Binding of 125I-labeled EGF (125I-EGF) to cultured fibroblasts results in a rapid loss of up to 90% of their specific binding capacity, which directly correlates with the degradation of the prebound hormone (3,4,6). EGF initially localized on the plasma membrane is translocated into cells by absorptive pinocytosis (7), after which the EGF-receptor complex is degraded (8, 9). The exact site of hormone degradation has yet to be determined but is most likely localized within lysosomes, because amines that inhibit lysosomal function, such as chloroquine, ammonium ion, and local anesthetics, prevent the degradation of 125I-EGF specifically bound to cells (3,4).In many instances, endocytosis of ligand-receptor complexes is preceded by surface aggregation of receptors into specialized regions of the plasma membrane (7,8,10,11 In this report, we show by direct biochemical methods that the process of EGF receptor internalization occurs even without EGF stimulation and is not prevented by the lysomotopic agents MeNH2 or chloroquine. These results, in sum, have important implications for understanding the normal processing of the EGF receptor and suggest a means to determine whether EGF-receptor processing is involved in mitogenic responses. MATERIALS AND METHODSMaterials and Cell Culture. Human fibroblasts from newborn foreskin (HF) and from an oral epidermal carcinoma (KB) were routinely cultivated as described (5). Mouse EGF was purified (17) from adult male submaxillary glands (Pel-Freez) and iodinated with chloramine-T (1) to a specific activity of 1.8 to 2.5 X 105 cpm/ng. 125I-EGF Binding Assay. Assays for HF cells were performed in monolayer cult...
Background & Aims Cholecystokinin (CCK) is a satiation peptide released during meals in response to lipid intake; it regulates pancreatic digestive enzymes that are required for absorption of nutrients. We proposed that mice with a disruption in the CCK gene (CCK-KO mice) that were fed a diet of 20% butter fat would have altered fat metabolism. Methods We used quantitative magnetic resonance imaging to determine body composition and monitored food intake of CCK-KO mice using an automated measurement system. Intestinal fat absorption and energy expenditure were determined using a noninvasive assessment of intestinal fat absorption and an open circuit calorimeter, respectively. Results After consuming a high-fat diet for 10 weeks, CCK-KO mice had reduced body weight gain and body fat mass and enlarged adipocytes, despite the same level of food intake as wild-type mice. CCK-KO mice also had defects in fat absorption, especially of long-chain saturated fatty acids, but pancreatic triglyceride lipase (PTL) did not appear to have a role in the fat malabsorption. Energy expenditure was higher in CCK-KO than wild-type mice and CCK-KO mice had greater oxidation of carbohydrates while on the high-fat diet. Plasma leptin levels in the CCK-KO mice fed the high-fat diet were markedly lower than in wild-type mice, although levels of insulin, gastric-inhibitory polypeptide, and glucagon-like peptide-1 were normal. Conclusion CCK is involved in regulating the metabolic rate and is important for lipid absorption and control of body weight in mice placed on a high-fat diet.
The stimulation of DNA synthesis by epidermal growth factor, insulin, and serum is inhibited by a variety of alkylamines when present for the duration of the stimulatory preincubation (20-24 hr). These results contradict an earlier report [Maxfield, F. R., Davies, P. J. A., Klempner, L., Willingham, M. C. & Proc. NatL Acadt Sci USA 76, [5731][5732][5733][5734][5735] and can be explained by differences in incubation conditions. The most straightforward interpretation of our results is that the mitogenic activities of growth factors are blocked by agents that inhibit the intracellular processing of hormone-receptor complexes. Therefore, the continued internalization and degradation of growth factors or their receptors within cells may play an important role in inducing mitogenesis in cultured human fibroblasts and may explain the prolonged requirement for epidermal growth factor in the culture medium (8 hr) to elicit a mitogenic response. We also found that bacitracin, a potent inhibitor of the enzyme transglutaminase, neither prevents receptor internalization or degradation in human fibroblasts nor inhibits the mitogenic activity of epidermal growth factor. These results suggest that transglutaminase activity may not be relevant to the mechanisms of growth-factor-induced receptor internalization or mitogenesis.A variety of serum proteins, including polypeptide hormones, are transported into cells by adsorptive pinocytosis directed by specific surface receptors (1, 2). Internalization of epidermal growth factor (EGF) is rapid and is mediated by a temperaturedependent process (1) that presumably takes place only within specialized coated regions of the plasma membrane (3, 4). The vesicles formed ultimately fuse with lysosomes, where the EGF (1, 5) and its receptor (6) are degraded. In light of these findings, it has been difficult to determine whether the long-term activities of peptide hormones, such as mitogenesis, are a consequence of a transduction mechanism solely through direct receptor binding (7) or are due to a translocation mechanism through the generation of an intracellular message via rapid internalization and processing of ligand-receptor complexes (8).Fluorescent derivatives of EGF, insulin, and a2-macroglobulin can be used to visualize surface receptors on cells (9-11).Initially, these labeled receptors are randomly dispersed on the cell surface, but they rapidly form surface aggregates, or patches, that ultimately are internalized. Studies with ferritin-EGF conjugates confirm this sequence of events (4,12). By fluorescent visual intensification microscopy, it was found that apparent receptor clustering and internalization requires calcium and is prevented by a variety of primary alkylamines; e.g., methylamine (MeNH2) and 1-dimethylaminonaphthalene-5-sulfonyl (dansyl) cadaverine (13). These amines are also known to inhibit the intracellular enzyme transglutaminase (14) and to indirectly inactivate lysosomal hydrolases, presumably by shifting the intralysomal pH (15). Furthermore, it has b...
acts peripherally as a satiating peptide released during meals in response to lipid feeding and centrally functions in the modulation of feeding, exploratory, and memory activities. The present study determined metabolic parameters, food intake, anxiety-like behaviors, and cognitive function in mice lacking the CCK gene. We studied intestinal fat absorption, body composition, and food intake of CCK knockout (CCK-KO) mice by using the noninvasive measurement of intestinal fat absorption along with quantitative magnetic resonance (QMR) imaging and the DietMax system, respectively. Additionally, exploratory and memory capacities were assessed by monitoring running wheel activity and conducting elevated plus-maze and Morris watermaze tests with these mice. Compared with wild-type (WT) littermate controls, CCK-KO mice had normal food intake, fat absorption, body weight, and body mass. CCK-KO mice ate more food than control animals during the light period and less food during the dark period. Energy expenditure was unchanged between the genotypes; however, CCK-KO mice displayed greater fatty acid oxidation. CCK-KO mice were as active as WT animals in the running wheel test. CCK-KO mice spent more time in the closed arms of an elevated plus-maze, indicative of increased anxiety. Additionally, CCK-KO mice exhibited attenuated performance in a passive avoidance task and impaired spatial memory in the Morris water maze test. We conclude that CCK is involved in metabolic rate and is important for memory and exploration. CCK is intimately involved in multiple processes related to cognitive function and food intake regulation. cholecystokinin 1 receptor; cholecystokinin 2 receptor; cognitive behaviors CCK OCCURS as a carboxyl terminally amidated peptide that exists in several possible lengths (43,58,66). In rats and mice, the predominant circulating forms include cholecystokinin octapeptide (CCK-8) and CCK-22, whereas larger molecular forms (CCK-33 and CCK-58) are also present in human and canine plasma (42,61,38,63,16). These molecular forms are processed from a 115-amino acid preprohormone product of the gene residing in chromosome 3 in rodents (13, 57). Intestinal endocrine cells secrete a mixture of medium-sized CCK forms, while central and peripheral neurons mainly release sulfated forms of 62). In response to consumption of either lipid and protein, CCK is produced and secreted by intestinal I cells (41). The release of CCK from neurons is caused by potassium ion-induced depolarization (15). Peripheral CCK is involved in modulating intestinal motility, stimulating pancreatic enzyme secretion, enhancing gallbladder contraction, and regulating meal size (11,21,24,29,48,56,64,69).CCK is also a neurotransmitter in many areas of the nervous system (6). Intraperitoneal or central administration of either purified CCK extracts or synthetic CCK-8 to fasted rats reduces food intake (22,33,37,54), and this is manifested by a reduction of meal size as opposed to reduced frequency of meals (22,37). Two types of CCK receptors (CCK1...
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