Cholesterol catabolism in the rabbit in fasted and fed states.

Urinary and fecal endogenous steroid excretion of fed or fasted New Zealand white rabbits was determined by the isotopic steady state method after subcutaneous implantation of radioactive cholesterol. While plasma cholesterol was increasing during a 9-day fast, fecal steroid excretion decreased to 10% of the excretion rates in the fed state. Refeeding the fasted rabbits led to a decrease in plasma cholesterol and an increase in fecal endogenous steroid excretion. Urinary steroid excretion, which represented 18% of total endogenous steroid excretion for fed animals, decreased during fasting and increased during refeeding, but these changes were relatively small. The small intestine, cecum, and colon of fed or fasted rabbits had similar endogenous steroid was acidic steroid. During attempts to alter the circulating bile acid concentration by supplying deoxycholate (200 mg/day) to fed rabbits or cholestyramine (2 g/day) to fasted rabbits, plasma cholesterol concentration did not change to the same extent as during fasting or refeeding, respectively. The decreased cholesterol catabolism and the hypercholesterolemia that are seen in the fasting rabbit may result from decreased clearance of plasma cholesterol.     

Studies of the regulation of purine nucleotide catabolism.

Ehrlich ascites tumor cells containing radioactive ATP were incubated in vitro with a range of concentrations of 2-deoxyglucose in order to produce different rates of ATP catabolism. Concentrations of all radioactive products of ATP catabolism were measured, and apparent rates of adenylate deaminase and inosinate dehydrogenase and of adenylate and inosinate dephosphorylation were calculated. It was concluded that these processes were reggulated primarily by the rate of formation of substrate, and to a lesser extent in some cases, by substrate concentration. No evidence was obtained for regulation of these processes by the concentration of ATP. The deoxyglucose-induced catabolism of radioactive GTP was also studied. When ATP catabolism was induced by incubation with 2,4-dinitrophenol, time courses of accumulation of purine nucleoside monophosphates and rates of alternative pathways of their metabolism were quite different than when deoxyglucose was used.     

Synthesis and catabolism of rabbit alpha 1-antitrypsins F and S.

The metabolic relationship between the two major forms of rabbit alpha 1-antitrypsin, F and S, was investigated by using labeling techniques in vivo and in vitro. After the injection of [14C]leucine, the S/F specific-radioactivity ratio showed characteristic changes with time: at 1 h, the ratio was high (1.2-1.4), but by later times (5-7h) it decreased to a value of approx. 1.1. Two different techniques were used to purify alpha 1-antitrypsin for labelling with iodine. The half-lives of the differentially labelled and simultaneously injected F- and S-forms were 68.1 (+/- 7.6 S.D) and 55.3 (+/- 8.1 S.D)h respectively. Combined electrophoretic and gamma-spectrometric studies provided no evidence for metabolic interconversion of the alpha 1-antitrypsin forms in the circulation. These observations suggest that rabbit alpha 1-antitrypsins F and S are, despite their close chemical composition and immunological identity, metabolically independent proteins. Therefore the possibility is raised that alpha 1-antitrypsin synthesis in rabbits is controlled by two autosomal genes or two sets of such genes.     

Studies on the regulation of leucine catabolism in the liver. Stimulation by pyruvate and dichloroacetate

The rate of oxidation of L-[1-14C]leucine to 14CO2 by isolated rat hepatocytes is increased by pyruvate and dichloroacetate. This effect is specific for L-leucine, not being observed for L-valine, L-isoleucine, or D-leucine. Transamination, the rate-limiting step of L-leucine catabolism in the liver, is the site of stimulation, because uptake of L-leucine by the cells and the oxidation of its transamination product, alpha-ketoisocaproate, are not increased. Measurement of steady state levels of alpha-ketoisocaproate indicate that both pyruvate and dichloroacetate promote the transamination of L-leucine, thereby increasing the availability of substrate for decarboxylation by the alpha-ketoisocaproate dehydrogenase complex (EC 1.2.4.3). Pyruvate stimulation of transamination is secondary to the provision of keto acid acceptors for the amino group of L-leucine. The mechanism of the effect of dichloroacetate remains unknown.     

Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes.

Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at O or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4 degrees C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.     

Receptor-mediated catabolism and tissue uptake of human low density lipoprotein in the cholesterol-fed, atherosclerotic rabbit

The LDL receptor pathway, which was delineated in cultured cells, is now known to operate in vivo. In this study we have measured the plasma clearances and tissue uptakes of native and chemically modified (1,2-cyclohexanedione-treated or reductively methylated) LDL in rabbits in order to determine the response of the pathway to a high-cholesterol diet. 1 week on the diet increased circulating LDL and suppressed its receptor-mediated plasma clearance and uptake into all tissues. The fractional catabolic rate of the lipoprotein via the receptor-independent route also fell. Continuation of the feeding program for 12 weeks accentuated these changes and virtually eliminated receptor uptake into all tissues so that the plasma decay curves of native and cyclohexanedione-treated LDL were superimposable. Lipoprotein assimilation by the aorta, however, did not follow this general trend. This tissue, after 12 weeks, was variably infiltrated by atheromatous deposits and the appearance of these lesions was associated with a substantial increase in the relative uptakes of both native and chemically modified (cyclohexanedione-treated and reductively methylated) LDL. We concluded (a) that expansion of tissue cholesterol pools virtually abolishes LDL receptor activity in rabbits; and (b) that LDL assimilation (both apparently receptor-mediated and receptor-independent) paradoxically increases at sites where the aorta is affected by atheromatous lesions.     

Studies on the role of the areA gene in the regulation of nitrogen catabolism in Aspergillus nidulans.

Mutants of Apergillus nidulans with lesions in a gene, areA (formerly called amdT), have been isolated by a variety of different selection methods. The areA mutants show a range of pleiotropic growth responses to a number of compounds as sole nitrogen sources, but are normal in utilization of carbon sources. The levels of two amidase enzymes as well as urease have been investigated in the mutants and have been shown to be affected by this gene. Most of the areA mutants have much lower amidase-specific activities when grown in ammonium-containing medium, compared with mycelium incubated in medium lacking a nitrogen source. Some of the areA mutants do not show derepression of urease upon relief of ammonium repression. The dominance relationships of areA alleles have been investigated in heterozygous diploids, and these studies lend support to the proposal that areA codes for a positively acting regulatory product. One of the new areA alleles is partially dominant to areA+ and areA102. This may be a result of negative complementation or indicate that areA has an additional negative regulatory function. Investigation of various amdR; areA double mutants has led to the conclusion that amdR and areA participate in independent regulatory circuits in the control of acetamide utilization. Studies on an amdRc; areA double mutant indicate that areA is involved in derepression of acetamidase upon relief of ammonium repression.     

Structural studies on proteoglycan catabolism in rabbit articular cartilage explant cultures

Mature rabbit articular cartilage cultures have been used to study the catabolism of aggregating proteoglycan monomers in normal cartilage. During the first 4 days of culture, about 40% of monomers are degraded and lose the ability to bind to hyaluronate. The non-aggregating products (NAgg-PG) have been isolated and compared structurally and immunologically to aggregating monomers (Agg-PG) purified from fresh tissue. The results show that: (1) NAgg-PG are smaller, more heterogeneous in size and have a lower protein/glycosaminoglycan ratio than Agg-PG. (2) NAgg-PG and Agg-PG have a very similar chondroitin sulfate/keratan sulfate ratio. (3) NAgg-PG have 25-50% lower disulfide content than Agg-PG. (4) NAgg-PG have only about 20% of the reactivity of Agg-PG towards a monoclonal antibody (12-20/1-C-6) specific for the hyaluronate binding region of the core protein. These results provide further evidence that proteoglycan catabolism in cartilage explants involves proteolysis of core protein resulting in separation of the hyaluronate binding region from the glycosaminoglycan-rich regions.     

A comparative study of the rate-limiting enzymes of cholesterol synthesis, esterification and catabolism in the rat and rabbit

1. The rat and rabbit are amongst the animal models most widely used in the study of human atherosclerosis, a disease state correlating with disturbances in cholesterol metabolism. 2. In order to relate the key regulatory enzymes of cholesterol synthesis, esterification and catabolism in the rat and rabbit to their differing degree of susceptibility to atherosclerosis, enzyme levels and their properties were determined in liver and intestine of both species. 3. Hepatic HMG CoA reductase and cholesterol 7 alpha-hydroxylase levels were significantly higher in the rat than in the rabbit, while intestinal HMG CoA reductase activity in the two species was comparable. Conversely, the capacity to esterify cholesterol as measured by ACAT activities was considerably greater in both sites in the rabbit compared to the rat. 4. The data suggest that differences in the key regulatory enzymes of cholesterol metabolism in both liver and intestine may reflect different methods of cholesterol utilization in the two species.     

Studies on the relationship between the molecular structure and the catabolism of insulin

The catabolism of insulins modified at the A1, B1 or B29 positions or containing a synthetic crosslink between the A1 and B29 positions has been studied in vivo and in vitro. The metabolic clearance rates (MCR) of insulin, proinsulin and chemically modified insulins have been measured by a priming-dose constant infusion technique in greyhounds. Insulins modified at A1 and B29, particularly the crosslinked materials, had markedly lowered MCR's whilst B1 analogues did not differ from insulin. Proinsulin and the A1-B29 crosslinked materials showed a markedly lowered degradability by glutathione-insulin transhydrogenase.     

Studies on the structure of the rabbit kidney brush border.

The effects of salts and non-ionic detergents on renal brush borders have been studied. 2 M sodium chloride, iodide or thiocyanate dissociated up to 40% of the protein from the brush borders, destroying the core filaments and resulting in the formation of membrane vesicles; EDTA had a similar effect on structure but released little protein. Triton X-100 and Nonidet P-40 extracted up to 60% of the protein including the major membrane glycoproteins and the enzymes trehalase, maltase and aminopeptidase (microsomal). Triton exhibited a selective effect on lipids removing phosphatidylserine, phosphatidylethanolamine and sphingomyelin but not the bulk of the phosphatidylcholine or cholesterol. The residual structures after Triton extraction comprised the core filaments associated with vesicles of lipid containing alkaline phosphatase and several other proteins. Treatment of these core-vesicle complexes with 2 M sodium chloride dissociated the filaments, releasing the vesicles which could be recovered as a pellicle on centrifugation. It is suggested that the proteins found in the vesicles might serve to interconnect the core filaments with the lipid bilayer.