Uptake of Tritiated Folates by Human Bone Marrow Cells in Vitro

S ummary . Using incubation periods up to 4 hr, it was demonstrated that uptake of tritiated pteroylglutamic acid ([³H]PteGlu) by human bone marrow cells in vitro was in the range of six-fold greater than uptake by reticulocytes. Uptake was temperature-dependent, increasing during 4 hr at 37°C but not at 4°C; a similar temperature dependence was found for the uptake of [³H]methyltetrahydrofolate ([³H-CH₃]H₄PteGlu). The pH optimum for [³H]PteGlu uptake was in the range of 7.4. Percentage uptake decreased as concentration of [³H]PteGlu increased. Preincubation with unlabelled PteGlu reduced uptake of subsequently added [³H]PteGlu by twice as much as did preincubation with methotrexate, suggesting that methotrexate may only partly share the uptake mechanism for [³H]PteGlu. [³H]PteGlu uptake was not affected by preincubation with diphenylhydantoin or a sulphydryl inhibitor. Uptake of [³H-CH₃]H₄PteGlu by human bone marrow cells appeared to be approximately twice the uptake of [³H]PteGlu. The findings support the concept of two mechanisms for folate uptake by human reticulocytes and bone marrow cells: an energy-dependent, active carrier mechanism probably of primary physiologic significance, and a seemingly passive diffusion-like mechanism, probably primarily of pharmacologic significance.     

Muscle growth and development in chick embryos--thyroidal influence on ribosomal RNA metabolism

Thyroidal influence on muscle ribosomal RNA (rRNA) metabolism was examined during late embryogenesis of the chick, when there is normally considerable growth. RNA polymerase I activity of leg muscle nuclei was 52% of the control value at 20 days of incubation, following 1 week of methimazole (MMI) treatment (P < 0.01). The enzyme activity for nuclei isolated from muscle of 19-day, MMI-treated embryos at 48 hr after the administraton of 300 ng of thyroxine (T₄) was 31% greater than for embryos receiving MMI alone (P < 0.01). At 24 hr after the administration of [¹⁴C]orotic acid, and 23.5 hr after the administration of 300 ng of T₄, the amount of ¹⁴C incorporated into the RNA from leg muscles of 18-day, MMI-treated embryos was 29% higher than for MMI-treated embryos receiving Ringer's solution at the time of hormone injection (P < 0.02); T₄ did not influence incorporation into total RNA at 3, 6, or 12 hr after administration of labeled precursor. The average specific activity of the cytoplasmic ribosomal-polysomal RNA fraction was increased approximately 43% in the T₄-treated embryos at 24 hr after administration of [¹⁴C]orotic acid, and 23.5 hr after hormone, compared to that of MMI controls (P < 0.01), and the specific activity of the combined 18-S and 28-S rRNA, isolated from the ribosomal-polysomal RNA fraction, was increased 39% in the T₄-treated embryos (P < 0.01). Data on the 24-hr, cumulative incorporation of [¹⁴C]orotic acid into perchloric acid-soluble components of leg muscle indicate that the increased incorporation of labeled precursor into RNA was not due to greater uptake of [¹⁴C]orotic acid into muscle or a higher specific activity of uridine nucleotide precursors. The present report of thyroidal influence on muscle rRNA metabolism supports earlier observations which indicated that thyroid function is important to muscle growth during late embryogenesis in the chick.     

Cobalamin Dependent Methionine Synthesis and Methyl-Folate-Trap in Human Vitamin B12 Deficiency

The activity of methionine synthetase (MS) is important for the rapid growth of human haematopoietic cells and cultured lymphoblastoid cells. The MS reaction is the only known metabolic step in which both vitamin B₁₂ and folate are essential in a single enzyme reaction. In vitamin B₁₂ deficiency the MS activity in bone marrow cells is significantly lower than that in normal bone marrow. Free tetrahydrofolic acid (H₄PteGlu) is normally liberated from its metabolically inactive storage form, 5-methyl-H₄PteGlu (CH₃H₄PteGlu), in the cobalamindependent MS reaction. Thus, in vitamin B₁₂ deficiency H₄PteGlu is not available in sufficient concentration to maintain the de novo synthesis of thymidylate and purines, and accords with the methyl-folate-trap hypothesis. After treatment with amethopterin (Methotrexate^TM), the incorporation of ³Hdeoxyuridine into cellular DNA is reduced. In proliferating normal cells this effect of methotrexate can be prevented (and the cells rescued) with CH₃-H₄PteGlu or with CHO-H₄PteGlu (5-formyl-H₄PteGlu; Leucovorin^TM). On the other hand, in vitamin B₁₂ deficient bone marrow cells this so-called rescue-effect could only be achieved with CHO-H₄PteGlu and not with CH₃-H₄PteGlu. These observations also support the hypothesis of the methyl-folate-trap in vitamin B₁₂ deficiency. Decreased MS activity in vitamin B₁₂ deficiency seems to be the essential metabolic fault, which is responsible for secondary alterations of folate metabolism. Thus, measurement of MS activity may allow direct functional assessment of vitamin B₁₂ deficiency, at least with regard to DNA metabolism.     

In Vitro Binding of Folates by Body Fluids

It is useful to differentiate between saturated folate binders in serum (carrying endogenous folate) and unsaturated binders (investigated in the present study). These two groups of binders need not necessarily be chemically identical and the unsaturated binder may even be an in vitro artifact, especially when measured with non-physiological folates. Macromolecular binding of radio-active N-5-methyl tetrahydrofolic acid (CH3H4PteGlu) and/or folic acid (PteGlu) by human serum and urine was assessed by means of exhaustive saline dialysis, haemoglobin-coated charcoal adsorption, column chromatography with DEAE-Sephadex A-50, and sucrose gradient analysis. Binding was found to be minimal or absent. Charcoal adsorption showed a mean serum binding capacity of 0 mug/1. for PteGlu and 0.58 mug/1. for CH3H4PteGlu. In pregnancy the mean serum values were 0.23 mug/1. for PteGlu, 0.66 mjg/1. for CH3H4PteGlu, and with folate deficiency 0.30 mug/1. for PteGlu, 0.49 mug/1. for CH3H4PteGlu. Mean urinary folate binding was minimal (less than 0.5 mug/1.), and red cell haemolysate similarly revealed very low binding on exhaustive dialysis. Column chromatography showed that tracer doses of [14C]PteGlu added to serum migrated distally to the protein zone; [14C]CH3H4PteGlu similarly showed no evidence of protein binding. On a sucrose gradient [14C]PteGlu also separated clear of the protein zone.     

Uptake of Vitamin B12 by Phytohaemagglutinin-Transformed Lymphocytes

Phytohaemagglutinin (PHA)-transformed lymphocytes have been used as a model cell system to study the uptake of radioactive vitamin B₁₂ by haemopoietic cells. Both mature granulocytes and PHA-transformed lymphocytes took up more vitamin B₁₂ than mature, non-transformed lymphocytes. Uptake of vitamin B₁₂ by PHA-stimulated lymphocytes was greatest at 48–72 hr of culture, i.e. at about the time or just before the time of peak DNA synthesis.
Vitamin B₁₂ deficient lymphocytes took up significantly less vitamin B₁₂ than normal lymphocytes. Folate deficient lymphocytes took up an average of about 50% more vitamin B₁₂ than normal but the difference was not statistically significant for the numbers tested. Vitamin B₁₂ uptake by PHA-stimulated lymphocytes was related to their rate of RNA synthesis (measured by ³H-uridine uptake) and was closely related to active cytoplasmic protein synthesis since it was rapidly inhibited by puromycin and cycloheximide.     

Lymphocyte Transformation in Megaloblastic Anaemia: Morphology and DNA Synthesis

S ummary . Lymphocytes from six patients with untreated pernicious anaemia and from one patient with folate deficient megaloblastic anaemia were cultured with phytohaemagglutinin (PHA). The mean percentage of cells undergoing transformation (78) was similar to normal. The morphology of the PHA-stimulated transformed 'megaloblastic'lymphocytes was distinct from that of PHA-stimulated normal lymphocytes. Transformed 'megaloblastic'lymphocytes tended to be larger and to show a more finely reticulated open chromatin pattern than normal transformed lymphocytes.
The incorporation of [³H]thymidine into DNA of PHA-stimulated 'megaloblastic'lymphocytes was significantly greater than normal. The inhibitory action of deoxyuridine (dU) on [³H]thymidine incorporation into the DNA of PHA-stimulated lymphocytes was significantly less in 'megaloblastic'lymphocyte cultures than in normal cultures. Both these observations suggest that there is a block in synthesis of thymine-DNA from dU in PHA-stimulated 'megaloblastic'lymphocytes.
The metabolic block in vitamin B₁₂ deficiency could be partly corrected by vitamin B₁₂ and completely corrected by folic acid or folinic acid suggesting that the defect was due to lack of folate coenzyme. The abnormality of thymine-DNA synthesis in 'megaloblastic'lymphocytes shown here was similar to that shown by other workers in megaloblastic bone marrow cells. The present study provides cytological and biochemical evidence that deficiency of vitamin B₁₂ or folate causes a derangement of DNA synthesis in the lymphocyte.     

Comparative studies of the intestinal absorption of [3H]pteroylmonoglutamate and [3H]pteroylheptaglutamate in man.

Comparative efficiencies of absorption of crystalline folic acid polyglutamate and monoglutamyl folic acid were determined in 11 normal subjects by measurement of the excretion of radioisotope in the urine after oral administration of [3H]pteroylheptaglutamic acid ([3H]PteGlu7) synthesized in our laboratory and of [3H]pteroylglutamic acid ([3H]PGA). Following ingestion of 0.6 mumole of [3H]PteGlu7, urinary excretion of radioactivity over 48 hr averaged 56.1 +/- 11.2% of the total dose. By comparison the ingestion of 0.6 mumole of [3H]PGA resulted in an average urinary excretion of 70.8 +/- 13.0% for the same time period. Approximately 90% of the urinary radioactivity was excreted during the initial 24-hr collection period. The mean recovery of radioactivity in urine and stool was 94% and recovery exceeded 84% in all subjects. The principal radioactive compound in the urine chromatographed with standard pteroylmonoglutamates. By chromatography, urinary folates were monoglutamates whether [3H]PGA or [3H]PteGlu7 was administered. The time course of folate absorption for the study compounds was compared by measuring the rise in serum radioactivity after the oral folate dose. Peak values in serum folate radioactivity following [3H]PGA occurred at 1 hr, whereas the peak values after [3H]PteGlu7 more often occurred at 2 hr. Only monoglutamyl folate was detected in the serum. These studies demonstrate that in normal subjects physiological doses of crystalline monoglutamyl and crystalline heptaglutamyl folates are both absorbed with high degrees of efficiency.     

The lymphocyte as a marker of past nutritional status: persistence of abnormal lymphocyte deoxyuridine (dU) suppression test and chromosomes in patients with past deficiency of folate and vitamin B12

In short-term suspension cultures of bone marrow cells or PHA-stimulated lymphocytes from normal subjects, non-radioactive deoxyuridine (dU) suppresses the incorporation of radioactive thymidine ([³H]TdR) or its analogue, [¹²⁵I]deoxyuridine ([¹²⁵I]Udr), into DNA. This normal suppression by deoxyuridine (dU) is impaired in both of these cell systems from patients with deficiency of folate or vitamin B₁₂, and corrected by the appropriate vitamin. Patients with megaloblastic anaemia due to deficiency of vitamin B₁₂ or folate were studied before and after treatment. When treatment had returned to normal the bone marrow morphology and the serum and red cell vitamin levels, then the dU suppression test and chromosomal changes in the bone marrow were also corrected. However, the dU suppression test and chromosomal changes remained abnormal in lymphocytes as long as 84 d after therapy. These abnormal lymphocyte dU suppression tests were corrected by the appropriate in vitro additions of folic acid, methylfolate and vitamin B₁₂, depending on the vitamin deficiency present before therapy. These studies suggest that an abnormal lymphocyte dU suppression test corrected by the appropriate vitamin in vitro, and characteristic chromosome abnormalities in lymphocytes, when these are absent in the bone marrow, indicate past deficiency of vitamin B₁₂ or folate. These changes can be used for retrospective diagnosis of these deficiencies in patients treated by ‘shotgun’ therapy. They further support the concepts that circulating unstimulated lymphocytes: (1) do not incorporate appreciable amounts of vitamin B₁₂ or folic acid; (2) reflect the vitamin status of the patient at the time the lymphocytes were generated; and (3) cannot replace bone marrow in dU suppression tests aimed at diagnosis of current marrow and other non-lymphocyte cell line nutrient status. These studies add to the evidence that selective nutrient deficiency may occur in one but not another cell line in the same person, and point to the need for more studies on factors affecting nutrient delivery, uptake, and utilization by various human cell lines. These studies also provide a new approach to evaluation of circulating lymphocyte age.     

Spontaneous synthesis and release of C16 juvenile hormone by isolated corpora allata of female locust Schistocerca gregaria and female cockroach Periplaneta americana.

Isolated pairs of corpora allata (CA) from adult females of both Schistocerca gregaria and Periplaneta americana incorporate radioactivity from [methyl-¹⁴C] methionine into C₁₆ juvenile hormone (JH) when incubated for up to several hours, such that the rate of JH synthesis can be accurately determined. Analysis of the medium shows that CA from P. americana continue to release newly synthesised JH at constant rates for at least 5 hr, whereas those from S. gregaria may show a marked decrease in rate of JH release after 3 hr, particularly in the case of glands having high initial rates of synthesis and release. In both cases the rates of release of JH are strictly proportional to the rates of JH synthesis when measured over a period of 3 hr incubation, independent of the rate of synthesis. It is concluded that the spontaneous level of endocrine activity in glands from both these species can be faithfully quantified by precise radiochemical methods using the short-term incubation procedures described.     

Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells

Aims/hypothesis The aim of this study was to determine whether adiponectin elicits glucose uptake via increased GLUT4 translocation and to investigate the metabolic fate of glucose in skeletal muscle cells treated with globular adiponectin.Materials and methods Basal and insulin-stimulated 2-deoxy-d-[³H]glucose uptake, cell surface myc-tagged GLUT4 content, production of ¹⁴CO₂ by oxidation of d-[U-¹⁴C]glucose and [1-¹⁴C]oleate, and incorporation of d-[U-¹⁴C]glucose into glycogen and lactate were measured in the presence and absence of globular adiponectin.Results RT-PCR and Western blot analysis revealed that L6 cells and rat skeletal muscle cells express AdipoR1 mRNA and protein. Globular adiponectin increased both GLUT4 translocation and glucose uptake by increasing the transport V_max of glucose without altering the K_m. Interestingly, the incorporation of d-[U-¹⁴C]glucose into glycogen under basal and insulin-stimulated conditions was significantly decreased by globular adiponectin, whereas lactate production was increased. Furthermore, globular adiponectin did not affect glucose oxidation, but enhanced phosphorylation of AMP kinase and acetyl-CoA carboxylase, and fatty acid oxidation.Conclusions/interpretation The present study is the first to show that globular adiponectin increases glucose uptake in skeletal muscle cells via GLUT4 translocation and subsequently reduces the rate of glycogen synthesis and shifts glucose metabolism toward lactate production. These effects are consistent with the increased phosphorylation of AMP kinase and acetyl-CoA carboxylase and oxidation of fatty acids induced by globular adiponectin.     

Immunosuppressive serum factors in viral hepatitis. I. Characterization of serum inhibition factor(s) as lymphocyte antiactivator(s)

Sera from patients with acute viral hepatitis B were found to inhibit the in vitro proliferation of normal lymphocytes induced by different mitogens and antigens. In addition, an effect on concanavalin A-induced T suppressor cell activity and pokeweed mitogen-stimulated IgG and IgM synthesis was demonstrated. Studies concerning the kinetics of serum immunosuppressive effects indicated that serum immunosuppressive factor (SIF) interfered with the intermediate phase of mitogen-induced lymphocyte activation which was defined by protein and RNA synthesis. Thus, when SIF-positive sera were added to lymphocytes, which were already activated by phytohemagglutinin, for 8, 12, or 18 hr, the inhibitory effect decreased in relation to the duration of lymphocyte activation. No inhibition could be demonstrated when SIF-positive sera were added 24 hr after initiation of mitogen stimulation. Furthermore, similar inhibitory effects were found measuring either uptake of [3H]uridine (RNA synthesis) or [3H]leucine (protein synthesis) in a 24 hr culture of phytohemagglutinin-stimulated lymphocytes or [3H]thymidine uptake (DNA synthesis) after 48 hr. These results indicate that SIF act(s) like an antiactivator and may belong to immunoregulatory physiologic serum factors.     

Transport of Methotrexate into Normal Haemopoietic Cells and into Leukaemic Cells and its Effects on DNA Synthesis

Summary . Methotrexate uptake by, and action on normal and leukaemic haemopoietic cells has been studied. Peak uptake occurred just prior to peak DNA synthesis, at 48 hr for normal, and at 6–7 days for chronic lymphocytic leukaemic lymphocytes stimulated by phytohaemagglutinin (PHA). Uptake of methotrexate into these cells and into normal bone marrow cells was consistently inhibited by dibutyryl cyclic 3′,5’adenosine monophosphate (dibutyryl cyclic AMP) at concentrations of 10^?3 to 10^?5m and by inhibitors of phosphodiesterase (theophylline, puromycin aminonucleoside and puromycin). Lower external concentrations of dibutyryl cyclic AMP (10^?5 to 10^?6m ), however, slightly increased the uptake of methotrexate. Dibutyryl cyclic AMP and the phosphodiesterase inhibitors at high concentrations also inhibited cellular uptake of 5-methyltetrahydrofolate and of thymidine into DNA. Conversely, none of these compounds inhibited uptake of pteroylglutamic acid (folic acid). These results suggest that the rate of entry of methotrexate and reduced folates (but not of folic acid) into haemopoietic cells may be governed by the intracellular concentration of cyclic AMP and that uptake of methotrexate by cells is related to uptake of natural folates but not to that of folic acid. Methotrexate uptake was also rapidly inhibited by actinomycin D suggesting that it is closely coupled to DNA synthesis. Rate of entry of methotrexate into acute leukaemic blast cells was roughly proportional to their rate of proliferation, measured by ³H-thymidine (³H-TdR) incorporated into DNA. Resistant cells were not found to show a defect in methotrexate transport in relation to their degree of proliferation. Methotrexate was found to inhibit mitosis, but not transformation of PHA-stimulated lymphocytes and this was presumably due to blocking of de novo thymidylate synthesis from deoxyuridylate, as shown by inhibition of incorporation of tritiated deoxyuridine (³H-dU) into DNA. Studies on human leukaemic blast cells showed differences between sensitive and resistant cases in the effect of methotrexate in vitro on ³H-dU and ³H-TdR incorporation into DNA and on dU blocking of ³H-TdR incorporation into DNA but none of these tests gave reliable prediction of resistance or sensitivity to methotrexate.     

Relationship between sterol synthesis and DNA synthesis in phytohemagglutinin-stimulated mouse lymphocytes.

Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. Rates of incorporation of radioactivity from [14-C]acetate into fatty acids and into CO2 by phytohemagglutinin-treated blood were not significantly different from control values. Phytohemagglutinin stimulation of sterol synthesis could be abolished by the addition of certain oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol and 20alpha-hydroxycholesterol) which specifically depress the activity of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl CoA reductase [mevalonate:NADP-nOXIDOREDUCTASE (CoA acylating); EC 1.1.1.34]. This treatment also abolished DNA synthesis and blastogenesis which otherwise followed the peak of sterol synthesis. Furthermore, DNA synthesis was repressed only if the inhibitor was added early enough to prevent sterol synthesis from reaching its usual maximum. When the compound was added after the rate of sterol synthesis had reached its maximum, DNA synthesis was not affected. These findings suggest that the synthesis of cholesterol is an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes after phytohemagglutinin activation.     

Regulation of isoproterenol-stimulated DNA synthesis in rat salivary gland in vivo by adrenal glucocorticoids

The magnitude of isoproterenol-stimulated DNA synthesis in rat liver submandibular gland varies 20-fold, depending on the time of day of drug injection, although diurnal regulation is not altered by aging. The diurnal response to isoproterenol reflects largely the hour to hour nutritional status of the rats as indicated by the inability of the drug to stimulate DNA synthesis in rats deprived of food for as little as 18 hr.Administration of glucocorticoids whose serum levels increase during fasting, in dosages as low as 0·05 mg per 100 g of body weight, both delays and reduces the magnitude of isoproterenol-stimulated DNA synthesis in rat submandibular gland. Inhibitory action of cortisol is effective when administered up to 8 hr following isoproterenol injection, and may be related to the delay in an Actinomycin D-sensitive event, required for DNA synthesis, which normally is stimulated at about that time. That isoproterenol-stimulated DNA synthesis may be regulated in vivo by endogenous glucocorticoids is indicated by: (1) acceleration and increased magnitude of isoproterenol-stimulated DNA synthesis in adrenalectomized rats; (2) inhibitory effects of corticosterone, the predominant glucocorticoid in rat, similar to those of cortisol; (3) specific binding of [³H]-corticol, [³H]-corticosterone and [³H]-dexamethasone to macromolecules derived from cytoplasmic extracts of submandibular gland; and (4) reduced uptake of [³H]-steroid following injection of isoproterenol.     

Synthesis and degradation of methylated proteins of mouse organs: Correlation with protein synthesis and degradation

L-(Methyl-¹⁴C)-methionine was administered i.p. to mice, and the incorporation of radioactive methionine into proteins and methyllysine and methylarginine residues formed by the transfer of the methyl-¹⁴C group of methionine were measured.Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with the protein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a similar degree. Neither the formation of S-adenosyl-(methyl-¹⁴C)-methionine nor protein methylase was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation.Radioactive methionine and the [C¹⁴] methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.     

In vitro glucose metabolism and lipid biosynthesis in bovine ventricular valves

In vitro oxygen uptake, pathways of [U-¹⁴C] glucose degradation and lipogenesis in the mitral and tricuspid valve of normal bovine heart were compared. In the mitral valve the oxygen consumption, ATP/ADP ratio, lipid content and the amounts of [¹⁴C]-glucose and [¹⁴C]-acetate incorporated into total lipids per mg DNA were higher, while lactate production and NADH/NAD ratio were lower, than those found in tricuspid valve. Triglycerides and phospholipids represented the largest lipid fractions in the mitral and tricuspid valve, respectively, and contained about 60% of the radioactivity incorporated into total lipids, but the highest relative rate of synthesis in the mitral and tricuspid valve was calculated to be for phospholipids and triglycerides, respectively. The bulk (94 to 98%) of [¹⁴C]-glucose incorprated into phospholipids and triglycerides by ventricular valves was found in the glycerol moiety. Incorporation of [¹⁴C]-glucose into fatty acids was markedly lower in the tricuspid than in the mitral valve. In the mitral valve the synthesis of 16 : 0 fatty acid from [I-¹⁴C] acetate proceeded de novo while in the tricuspid valve by a combination of de novo synthesis and chain elongation.     

Cellular cholesterol regulation — A defect in the Type 2 (non-insulin-dependent) diabetic patient in poor metabolic control

Summary This study investigates the relationship between Type 2 (non-insulin-dependent) diabetes mellitus and hypercholesterolaemia with regard to delivery of cholesterol to cells and regulation of endogenous cholesterol synthesis. The ability of LDL, from hypercholesterolaemic and Type 2 diabetic patients, to suppress cellular cholesterologenesis and to enhance mitogen-stimulated lymphocyte proliferation was compared. Cholesterol synthesis was estimated by measuring [¹⁴C]-acetate incorporation into cholesterol and lymphocyte proliferation was assessed by [³H]-thymidine incorporation into mitogen-stimulated normal lymphocytes. The results indicate that LDL from both Type 2 diabetic patients in poor metabolic control and hypercholesterolaemic patients was significantly less effective (p < 0.001) than LDL from non-diabetic normocholesterolaemic subjects in suppressing cholesterol synthesis in lymphocytes. LDL from all hypercholesterolaemic patients enhanced lymphocyte proliferation to a greater extent than LDL from normocholesterolaemic subjects and this effect was significantly increased using LDL from Type 2 diabetic, hypercholesterolaemic patients. Both suppression of [¹⁴C]-acetate incorporation and enhancement of [³H]-thymidine uptake could be related to an increased esterified/free cholesterol ratio in the LDL particle. The fact that cholesterol synthesis and cell proliferation were markedly altered by the above changes in LDL composition suggests a mechanism for cellular cholesterol accumulation in the Type 2 diabetic patient, even in the absence of elevated serum cholesterol levels.     

An Alteration in Glucose Metabolism Associated with a Defect in Ketone Body Metabolism

Skin fibroblasts (CC-69) cultured from a patient with a unique syndrome of ketoacidosis associated with coenzyme A transferase (EC 2.8.3.5) deficiency showed an altered pattern of carbohydrate metabolism. These cells used glucose at a rate significantly less than controls (125 against 680 nmol/mg per hr). The oxidation of [6-¹⁴C]glucose to ¹⁴CO₂ by these cells was also significantly diminished (12 against 350 pmol/mg per hr), but [2-¹⁴C]pyruvate and [1,4-¹⁴C]succinate oxidation by these cells did not differ from that by control cells. Measurements of glycolytic intermediates showed a reduction of several intermediates in the CC-69 cells that confirmed an inhibition of glycolysis between fructose-1,6-bisphosphate and pyruvate. The apparent inhibition in these cells could be reversed by an extended incubation of the cells in a buffered glucose solution. After 18 hr of incubation in 2.5 mM glucose, glucose uptake by the CC-69 cells increased 20-fold to 2560 nmol/mg per hr, whereas the rate for control cells remained constant at 640 ± 90. Concomitant with this increase, [6-¹⁴C]glucose oxidation rose from 8 to 2261 pmol/mg per hr while controls remained constant at 428 ± 175. This change was not due to new enzyme formation because incubation with puromycin had no effect on the increased use of glucose. Mixing experiments demonstrated no transfer of a permeable inhibitor or activating substances. In view of the deficiency of coenzyme A transferase in these cells, the data suggest an indirect regulatory role for this enzyme in peripheral tissue glycolysis.     

The role of the enterohepatic cycle in folate supply to tumour in rats

The importance of the folate enterohepatic cycle in governing the supply of folate to implants of a rapidly-growing tumour were studied in a new animal model. Following enteric administration of tritiated pteroylglutamic acid, [3H]PteGlu1, tumour uptake of labelled folate was limited to CH3[3H]H4PteGlu1 produced by the gut mucosal cells during absorption or subsequently recirculated through the enterohepatic cycle. 50% of the labelled folate reaching the tumour nodules in the first 6 h after enteric administration first circulated through the enterohepatic cycle. In addition, labelled folate taken up by tumour was immediately incorporated into a polyglutamyl folate pool. There was no evidence for a release of labelled folate from tumour for recirculation to the liver. Therefore the liver and folate enterohepatic cycle appear to play a major role in regulating the supply of folate to rapidly proliferating tissues such as tumour by acutely storing folate from the diet and then secreting it into bile for reabsorption and transport to tissue.