Vitamin B6 metabolism in Morris hepatomas

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.     

Determination of nonspecific lipid transfer protein in rat tissues and Morris hepatomas by enzyme immunoassay

Rat tissues contain a nonspecific transfer protein which in vitro mediates the transfer of diacylphospholipids as well as cholesterol between membranes. This protein appears identical to sterol carrier protein. A specific enzyme immunoassay for this protein was developed using antibodies raised in rabbits, against a homogeneous protein from rat liver. This assay was based on the very high affinity of the nonspecific lipid transfer protein for polyvinyl surfaces. A reproducible adsorption was achieved by presenting the protein to the surface in the presence of a large excess of bovine serum albumin. The adsorbed protein was detected with specific immunoglobulin (IgG) isolated by antigen-linked affinity chromatography and a goat anti-rabbit IgG-enzyme conjugate. Adsorption was proportional to the amount of protein present, giving rise to a linear standard curve. The enzyme immunoassay measured transfer protein levels in the range 0.2-2 ng. The highest concentrations of transfer protein were found in liver and intestinal mucosa. Levels in other tissues including brain, lung, kidney, spleen, heart, adrenals, ovary and testis were 5-10-fold lower than in liver. In the fast-growing Morris hepatoma 7777 the concentration of nonspecific lipid transfer protein was approximately one-tenth of that measured in the host liver, whereas a reduction of 65% was observed in the slow-growing Morris hepatomas 7787 and 9633. Subcellular distribution studies showed that approx. 70% of the transfer protein was present in the soluble supernatant fraction.     

Possible role for intracellular cholesteryl ester transfer protein in adipocyte lipid metabolism and storage

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.     

Cyclic AMP phosphodiesterase activity in three Morris hepatomas.

Rat liver cAMP phosphodiesterase has been fractionated into four peaks of activity with isoelectrofocusing column chromatography. The major two liver peaks (high Km enzymes) decreased with increasing growth rate while the minor two liver peaks (low Km enzymes) increased in one fast growing Morris hepatoma. There was also less total phosphodiesterase activity in the fast growing hepatoma.     

Regulation of rat liver phosphofructokinase levels in Morris hepatomas.

The levels of the major liver phosphofructokinase isozyme (PFK-L₂) and the PFK regulatory factor were measured in adult and fetal liver as well as Morris hepatomas of different differentiation states. The results indicate that both the PFK-L₂ activity and the PFK regulatory factor levels in well and highly differentiated hepatomas are not statistically different from the amounts found in adult liver. In the poorly differentiated hepatomas and fetal liver, the levels of both PFK-L₂ and PFK regulatory factor are approximately 2 and 3 fold greater, respectively, than what was found in adult liver.     

Hepatic lipid metabolism: effect of spermine, albumin, and Z protein on microsomal lipid formation.

Effects of spermine, bovine serum albumin, and Z protein on microsomal lipid formation from sn-glycerol 3-phosphate and [¹⁴C]palmitoyl CoA were investigated. In the presence of these agents, microsomal lipid formation was stimulated. This was attributed to the activation of sn-glycerol 3-phosphate acyltransferase and to the inhibition of palmitoyl CoA hydrolase. In addition to palmitoyl CoA, spermine also reacted with microsomal membranes in causing their aggregation, and ATP reversed the effect of spermine. Further studies indicated that the interaction of spermine with palmitoyl CoA, rather than with microsomal membranes, was responsible for the activation of glycerolipid formation or to the inhibition of palmitoyl CoA reductase. Examination of the intravesicular distribution of sn-glycerol 3-phosphate acyltransferase and palmitoyl CoA hydrolase and the effects of structural integrity of microsomal vesicles on these two membrane-bound enzymes indicated that the activation of glycerolipid formation and the inhibition of palmitoyl CoA hydrolase by spermine, bovine serum albumin, or Z protein may be closely linked with the structural integrity of microsomal vesicles.     

The role of CD36 in the regulation of myocardial lipid metabolism

Since the heart has one of the highest energy requirements of all organs in the body, it requires a constant and plentiful supply of fuel to function properly. Mitochondrial oxidation of lipids provides a major source of ATP for the heart, and the cellular processes that regulate lipid uptake and utilization are important contributors to maintaining proper myocardial energetic status. Although numerous proteins are coordinately regulated in order to ensure proper fatty acid utilization in the cardiomyocyte, a key first step in this process is the entry of fatty acids into the cell. An important protein involved in the transport of fatty acids into the cardiomyocyte is the plasma membrane-associated protein known as fatty acid translocase (FAT; also known as CD36). While multiple proteins are involved in facilitating fatty acid uptake in the heart, CD36 accounts for approximately 50–70% of the total fatty acid taken up in cardiomyocytes. As such, myocardial metabolism of fatty acids may depend upon proper CD36 function. Consistent with this, changes in CD36 levels/function have been implicated in the alteration of myocardial metabolism in the pathophysiology of certain cardiovascular diseases. As such, a better understanding of the role and function of CD36 in the heart may provide important insights for the development of new treatments for specific cardiovascular diseases. Herein, we review the role of CD36 in myocardial lipid metabolism in the healthy heart and describe how CD36-mediated alterations in lipid metabolism may contribute to cardiovascular disease. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

The role of ubiquinones in the regulation of lipid metabolism in rat thymocytes

The effect of ubiquinones Q-1, Q-2, Q-8 and Q-9 on lipid metabolism in rat thymocytes in vitro was studied. The cells were incubated in a medium containing ubiquinones within the concentration range from 1 to 100 microM. A 2-fold decreased cholesterol synthesis was observed in thymocytes incubated with ubiquinone Q-9 at a concentration of exogenous ubiquinone of no less than 40 microM. Incubation of thymocytes with ubiquinones UQ-1 and UQ-2 that are characteristic of rats (40 microM and 100 microM) resulted in a decrease of cholesterol synthesis. Ubiquinone-8 had a tendency to inhibit the cholesterogenesis in rat thymocytes.     

Activity of chromatin-bound protease from rat liver and Morris hepatomas.

1. The activity of serine protease was studied in chromatin and in histone preparations from rat liver and Morris hepatomas, lines 5123 D and 7777. 2. Electrophoretic patterns of histones and the amounts of acid-soluble peptides released during incubation of chromatin and histones showed that there was no significant correlation between protease activity and tumour differentiation and its growth rate.     

Serum lipoproteins in rats bearing Morris hepatomas of different degrees of differentiation.

Serum lipoproteins were measured by ultracentrifugal means in rats bearing hepatomas of different degrees of malignancy (Morris hepatomas 16, 5123TC and 7777) to determine the effect of these hepatomas on serum lipoprotein levels. Serum lipoprotein patterns were altered, especially in rats bearing hepatomas 16 and 7777, which had elevated high-density lipoproteins. (They were not elevated in serum of rats bearing hepatoma 5123TC). This increase in high-density lipoproteins seems to be specific for chemically induced hepatomas since HDL2 is usually decreased in humans and animals with types of cancer not involving the liver. It appears that hepatomas can synthesize lipoproteins, and the serum levels of the host rats are altered depending on the hepatoma. Different biochemistries appear to be associated with each hepatoma. Cholesterol and fatty acid levels of unfractionated serum and of isolated lipoproteins also indicate abnormal lipid/lipoprotein metabolism associated with these hepatomas.     

Phospholipid transfer activities in Morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas of 9633 and 7777, and compared to the activities found in normal and host liver. A 2-3-fold increase was found in the phosphatidylcholine and phosphatidylinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed. The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid o f a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously.     

Properties of guanylate cyclase in adult rat liver and several Morris hepatomas.

Guanylate cyclase (GTP pyrophyosphate-lyase (cyclizing), EC 4.6.1.2) activity was examined in preparations from normal rat liver and a series of Morris hepatomas. Homogenate gyanylate cyclase activites were 3.2, 1.6 and 1.2 nmol cyclic GMP formed per min/g tissue ihe non-substrate analogs of IMP were weak inhibitors of this enzyme, GMP and four of its analogs had Ki values ranging from 30 to 80 muM. The GMP analogs (8-azaGMP, 7-deaza-8-azaGMP, 2'-dGMP and beta-D-arabinosylGMP) and GMP were competitive inhibitors with respect to GTP.     

The role of protein translocation in the regulation of glycogen metabolism

Early biochemical analyses of metabolic pathways assumed that the free diffusion of substrates and enzymes in an evenly mixed cellular space provided the interactions that enabled reactions to proceed. Metabolic complexes have since been shown to assemble and disassemble in response to changes in cellular conditions, and in turn, to channel metabolic intermediates within discreet cellular compartments, allowing for the efficient use or storage of energy. A fundamental component to the formation of metabolic complexes and the channeling of metabolites is the translocation of enzymes in response to specific extra- and intracellular signals. These generalities play an important role in the metabolism of glucose to glycogen within skeletal muscle and liver. In this review, the similarities and differences in skeletal muscle and liver glucose metabolism with regards to glucose transport and intracellular processing will be addressed during the fasted to fed transition. More specifically, the importance of isoform expression and protein translocation in the tissue specific control of glucose homeostasis will be covered.     

Possible role of prolactin in growth regulation

To study the possible role of prolactin on growth regulation pituitary grafted rats of different ages and their sham-operated controls have been used. After the transplant operation of one pituitary gland from a litter-mate donor on day 5 or on day 30 of life, a marked prolactin increase has been observed. This increase has been immediate in 30 day-old rats and delayed in 5 day-old rats in which the elevation starts being significant on day 20 for females and on day 25 for male rats. Pituitary grafting on day 5 of life, with an adult donor gland, resulted in an immediate and marked increase of prolactin values in both sexes. Litter-mate donor pituitary grafting, on day 5 of life, resulted in an increase in body weight that could be directly correlated with the increase in prolactin levels. Adult pituitary grafting resulted in an increased body weight in females with no effects being detected in males. In 30 day-old grafted male and female rats, marked body weight increases were seen, over the whole studied period, together with an increase in nose-tail length (1 cm in female and 1.5 cm in males longer than the control animals). All these changes do not seem not be related to GH modifications in pituitary grafted rats, since GH changes were very slight with a final tendency to lower values in female rats but not in males. All these data could suggest that prolactin might exert a direct effect on growth both in male and female rats.(ABSTRACT TRUNCATED AT 250 WORDS)