Effect of fat and microflora on hepatic,small intestinal and colonic HMG CoA reductase,cytochrome P450 and cytochrome B5

High levels of dietary fat caused a significant reduction in HMG CoA reductase activity in the liver of germ-free rats whereas significantly elevated small intestinal enzyme activity was observed. Dietary fat had no significant effect on HMG CoA reductase activity in any tissue studied in the conventional rat. No significant change in colonic HMG CoA reductase activity was observed between any of the experimental groups. Rats fed a high-fat diet tended to exhibit higher cytochrome P₄₅₀ levels in all tissues studied, regardless of the presence of intestinal microflora.     

Regulation of 3-hydroxy-3-methylglutaryl CoA reductase by analogs of cholesterol and bile acids in cultured intestinal mucosa

Sodium fusidate and its glycine conjugate, which have the same detergent properties as bile acids, significantly (p<0.05) stimulate HMG-CoA reductase of cultured intestine below the critical micellar concentration (CMC) without affecting brush border enzymes. Above CMC, both amphiphiles are cytotoxic. At concentrations between 1 and 5 mM, sodium fusidate decreased cholesterol contents of cultured mucosa (p<0.05), the increase in synthesis only partially compensating for the sterol loss. Oxygenated sterols, 7-keto- and 25-hydroxycholesterol, also depleted mucosal cholesterol at 0.5 mM, exerting their effect differently by inhibiting HMG-CoA reductase (p<0.01). In contrast to their marked effect on total mucosal cholesterol contents, brush border cholesterol was unaffected by both cholesterol and bile acid analogs.     

A Synthetic mirror image of kalata B1 reveals that cyclotide activity is independent of a protein receptor

Featuring a circular, knotted structure and diverse bioactivities, cyclotides are a fascinating family of peptides that have inspired applications in drug design. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anti-cancer, anti-HIV, and hemolytic activities. In all of these activities, cell membranes appear to play an important role. However, the question of whether the activity of cyclotides depends on the recognition of chiral receptors or is primarily modulated by the lipid-bilayer environment has remained unknown. To determine the importance of lipid membranes on the activity of the prototypic cyclotide, kalata B1, we synthesized its all-D enantiomer and assessed its bioactivities. After the all-D enantiomer had been confirmed by (1)H NMR to be the structural mirror image of the native kalata B1, it was tested for anti-HIV activity, cytotoxicity, and hemolytic properties. The all-D peptide is active in these assays, albeit with less efficiency; this reveals that kalata B1 does not require chiral recognition to be active. The lower activity than the native peptide correlates with a lower affinity for phospholipid bilayers in model membranes. These results exclude a chiral receptor mechanism and support the idea that interaction with phospholipid membranes plays a role in the activity of kalata B1. In addition, studies with mixtures of L and D enantiomers of kalata B1 suggested that biological activity depends on peptide oligomerization at the membrane surface, which determines affinity for membranes by modulating the association-dissociation equilibrium.     

Non-insulin dependent diabetes mellitus in Psammomys obesus is independent of changes in tissue fatty acid composition

Recently it has been postulated that membrane fatty acid composition may be involved in the pathogenesis of insulin resistance and non-insulin dependent diabetes mellitus (NIDDM). The aim of this study was to determine whether alterations in tissue phospholipid (PL) fatty acids are present in hyperglycemic and hyperinsulinemic Psammomys obesus. On a native diet of salt bush, P. obesus (Israeli sand rat) remains lean and free of diabetes; however, when placed on a normal laboratory chow, a significant proportion of these animals develops a number of metabolic disorders associated with NIDDM, providing an ideal animal model of obesity and NIDDM. Four groups of mature P. obesus were studied: group A; normoglycemic and normoinsulinemic; group B: normoglycemic and hyperinsulinemic; group C: hyperglycemic and hyperinsulinemic; and group D: hyperglycemic hypoinsulinemic. In liver and red gastrocnemius muscle, there were no significant differences between groups, A, B, and in fatty acid composition of PL. Minor differences in individual fatty acids were demonstrated in group D animals (increased liver 20∶4n-6 and increased muscle 22∶5n-3); however, the unsaturation indices in liver and muscle were not significantly different between any of the groups. In considering that the minor changes in group D animals were not demonstrated in hyperinsulinemic group B animals or hyperglycemic, hyperinsulinemic group C animals, it is likely that the differences in group D animals were secondary to the more severe disturbances in glucose homeostasis and hypoinsulinemia present in these animals. The results of this study suggest that in this rodent diabetic model significant disturbances in glucose homeostasis and hyperinsulinemia may develop independently of changes in tissue fatty acid composition.     

Characterization of calcium-independent cytosolic phospholipase A2 activity in the submucosal regions of rat stomach and small intestine

This study was undertaken to compare the calcium-independent phospholipase A₂ (PLA₂) activities in the cytosols of twelve rat tissues and to determine whether their activities were distinct. 1-O-Alk-1′-enyl-2-[¹⁴C]-oleoyl-sn-glycero-3-phosphocho-line (PlsC) and 1-O-Alk-1′-enyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphethanolamine (PlsE) were synthesized and used as substrates, instead of phosphatidyl compounds, to exclude hydrolysis by cytosolic PLA₁ activity that could be present in some of the cytosolic preparations. For each tissue, we examined substrate specificity, pH optimum, and effect of adenosine triphosphate (ATP) and ATP analogues. PLA₂ activity was detected in eleven out of the twelve issues examined. Based on substrate specificity and pH optimum, cytosolic calcium-independent PLA₂ were classified in three groups. The first group, which included PLA₂ from small intestine, stomach and spleen, had the highest specific activity with PlsC as substrate (1253, 309 and 75 nmol/mg protein/hour, respectively) and an optimal pH at 6.5. Activity with PlsE as substrate was much lower (20–37%) than with PlsC. The second group of PLA₂ activities included the cytosolic activities from thymus, lung, liver and pancreas that showed lower specific activities for both substrates (14–23 nmol/mg protein/hour with PlsC) and had a broader optimal pH range of 6.1 to 7.5. The cytosols from brain, kidney, heart and muscle comprised the third PLA₂ group that was found to have a higher specific activity with PlsE (5–20 nmol/mg protein/hour) than PlsC and an optimal pH range from 7.4 to 7.9. Since the highest specific activity was found in the cytosol from small intestine, this PLA₂ was examined further. PLA₂ activity was found to be equally distributed in the cytosol of the submucosal portion of duodenum, jejunum and ileum with an optimal pH of 6.1 and a 5-fold higher activity with PlsC than PlsE as substrate. Moreover, this PLA₂ activity was inhibited by treatment with detergents. These results indicate the presence in the submucosal portion of the intestine of a calcium-independent cytosolic PLA₂ with a high specific activity toward PlsC and properties distinct from those described for the PLA₂ found in the intestinal brush-border.     

Perinatal development of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in rat lung,liver and brain

The developmental pattern of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), which catalyzes the rate-limiting step of cholesterol biosynthesis, was studied in lung, liver and brain of Sprague-Dawley rats. Each tissue exhibited a distinet pattern. Reductase activity in the fetal lung reached a peak at 19 days of gestation, which corresponds to the onset of active surfactant production. This observation is consistent with the suggestion that the fetal lung synthesizes all surfactant components including cholesterol. In the liver, reductase activity varied in a reciprocal fashion with serum cholesterol levels. The peak of brain reductase activity occurred at 3 days after birth at the onset of rapid brain growth despite rapidly rising serum cholesterol levels.     

Erythrocyte contamination of leukocyte populations following density-gradient centrifugation results in artificially high levels of human leukocyte HMG-CoA reductase activity

When measuring human leukocyte HMG-CoA reductase activity, special care must be taken to prevent erythrocyte contamination of the leukocyte layer during isopycnic centrifugation. Contamination during leukocyte isolation and subsequent erythrocyte lysis during NH₄Cl treatment results in increased leukocyte microsomal HMG-CoA reductase activity. Increased enzyme activity is not due to enzyme dephosphorylation, thiol-disulfide reduction or increased enzyme protein concentration. Leukocyte populations containing granulocytes appear to be most sensitive. Prevention of erythrocyte contamination during isopycnic centrifugation should aid in accurate measurement of human leukocyte HMG-CoA reductase activity.     

Abnormal suppression of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by hypertriglyceridemic very low density lipoprotein subclasses

Our previous studies showed that hypertriglyceridemic very low density lipoproteins (HTG VLDL) are functionally abnormal. HTG VLDL, but not normal VLDL, suppress HMG-CoA reductase in cultured normal human fibroblasts. To determine if the suppression by HTG VLDL resulted from a subpopulation of smaller suppressive particles, more homogeneous subclasses of VLDL-VLDL₁ (S_f 100–400), VLDL₂ (S_f 60–100), and VLDL₃ (S_f 20–60) were obtained from the d<1.006 (g°ml⁻¹) fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient and tested for suppression in normal human fibroblasts. VLDL₁ and VLDL₂ from each of the 12 normolipemic subjects tested failed to suppress HMG-CoA reductase activity in normal fibroblasts. Eleven out of 12 preparations of normal VLDL₃ suppressed HMG-CoA reductase, but only one-third as effectively as LDL. By contrast, the VLDL₁, VLDL₂ and VLDL₃ from 15 out of 17 hypertriglyceridemic patients (hyperlipoproteinemia Types IIb, III, IV and V) were highly effective in suppression, with half-maximal suppression at 0.1–2.0 μg VLDL protein/ml. The VLDL abnormality is apparently associated with hypertriglyceridemia and not hypercholesterolemia, since VLDL from a homozygous familial hypercholesterolemia patient with a Type IIa pattern did not suppress whereas each of the VLDL subclasses from a Type IIb patient suppressed. Suppression by HTG VLDL in normal cells is apparently a consequence of interaction of the protein portion of the VLDL with the specific LDL cell surface receptor since HTG VLDL₁ treated with 0.1 M 1,2-cyclohexanedione to block arginyl residues failed to suppress the enzyme. Moreover, hypertriglyceridemic S_f 60–400 VLDL failed to suppress HMG-CoA reductase activity in LDL receptor-negative fibroblasts. There were no consistent major compositional differences between comparable normal and hypertriglyceridemic VLDL subclasses which could account for differences in suppression. All VLDL subclasses from Type III subjects were enriched in cholesteryl esters and depleted in triglyceride, relative to the corresponding normal VLDL subclasses. However, Type IV and Type V VLDL subclasses were normal in this repect. We conclude from these studies that small particle diameter is not required for suppression, since HTG VLDL₁ and VLDL₂ which contained few, if any, small particles were effective in suppression. Presented as part of the symposium “Low Density and Very Low Density Lipoproteins” at the American Oil Chemists' Society meeting on May 2, 1979, in San Francisco.     

Effect of serum lipoproteins of bile obstructed rats on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in perfused rat liver

Total lipoproteins as well as fractionated VLDL+LDL and HDL from fasted control rats and bileligated rats were tested in liver perfusion for their effect on 3-hydroxy-3-methylglutaryl CoA reductase to increase 3-hydroxy-3-methylglutaryl CoA reductase activity than that of the control total lipoproteins. When the fractionated lipoproteins were tested from fasted control rats, it was found that the major stimulating activity was in the HDL fraction with minor activity in the VLDL+LDL fraction. When these plasma components isolated from fasted bile-ligated rats were tested, it was found that the major activity had shifted to the VLDL+LDL fraction with the HDL having only a minor stimulatory role. The possible mechanism of action of the abnormal lipoproteins associated with bile obstruction in regulating 3-hydroxy-3-methylglutaryl CoA reductase activity is discussed.     

The effects of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition on tissue levels of carnitine and carnitine acyltransferase activity in the rabbit

Recently, a new class of lipid lowering agents [3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors] was introduced into clinical practice. The use of these agents could lead to a secondary deficiency in carnitine, which may manifest clinically as a myalgia/myositis—a side effect that is occasionally seen with this class of drugs. In the present study, we examined the effect of an HMG-CoA reductase inhibitor (lovastatin) on serum and tissue levels of carnitine and carnitine acyltransferase activities in the rabbit. Rabbits (n=6) were fed chow containing lovastatin (30 mg/d) for 16 wk. Blood was collected and tissues (liver, heart, and skeletal muscle) harvested at sacrifice. Free and total carnitine were measured in serum and tissues by a radioenzymatic method. Carnitine acetyltransferase and carnitine palmitoyltransferase (CPT) activities were determined and expressed relative to DNA. Serum free (24.0±2.6 vs. 29.4±3.1 μM) and total (35.1±4.7 vs. 52.8±8.8 μM) carnitine levels increased significantly with 16 wk of treatment. This increase in total carnitine was mainly due to an increase in the levels of serum acylcarnitine (12.7±3.1 vs 26.5±5.7 μM). Tissue levels of total carnitine were significantly decreased by the treatment. Carnitine acetyltransferase was unaffected by the treatment, whereas there was a significant increase in the activity of CPT in the liver and heart.     

A novel bis-aziridinylnaphthoquinone with anti-solid tumor activity in which induced apoptosis is associated with altered expression of Bcl-2 protein

Aziridine-containing compounds have been of interest as anticancer agents since the late 1970s. The design, synthesis, and study of aziridinylnaphthoquinone analogues to obtain compounds with enhanced activity/toxicity profiles are an ongoing research effort in our group. A series of bis-aziridinylnaphthoquinone derivatives has been prepared, and the cytotoxic activities of these synthetic bis-aziridinylnaphthoquinone derivatives has been investigated. The synthetic derivatives displayed significant cytotoxicity against human carcinoma cell lines and weak cytotoxic activities against skin fibroblasts (SF). The bis-aziridinylnaphthoquinone 1 c was the most effective of the tested analogues at reducing the viability of Hep2 cells, with an LD(50) value of 5.23 microM, and also exhibited weak cytotoxic activity against SF cells, with an LD(50) value of 54.12 microM. The DNA alkylation and DNA interstrand cross-linking abilities of 1 c were also investigated. Bis-aziridinylnaphthoquinone 1 c was an effective agent for alkylation of DNA after chemical reduction in vitro, and its bifunctional alkylating moieties were able to cross-link DNA. We also report here our efforts to determine direct antitumor effects of 1 c on Hep2 cells. Growth arrest in Hep2 cells was preceded by early induction of G(2)-M cell cycle arrest at 0.75 microM of 1 c after culture for 24 h, and was then followed by apoptosis after 60 h. This was associated with decreased expression of antiapoptotic bcl2 protein (by 78 %) upon culture with 3.0 microM of 1 c after 60 h. Our results suggest that 1 c is a novel antitumor aziridinylnaphthoquinone with therapeutic potential against solid tumors.