Maria Rosa Lovati

Effects of sub-chronic exposure to SO2 on lipid and carbohydrate metabolism in rats

Sulfur dioxide (SO2) is a ubiquitous air pollutant, present in low concentrations in the urban air, and in higher concentrations in the working environment. While toxicological reports on SO2 have extensively dealt with the pulmonary system, essentially no data are available on the effects of chronic exposure to this pollutant on intermediary metabolism, although some biochemical changes in lipid metabolism have been detected. The present investigation was aimed at evaluating the effects of sub-chronic exposure to SO2 on concentrations of serum lipids/lipoproteins and on glucose metabolism, in animal models of hypercholesterolemia and diabetes. A specially designed controlinert atmosphere chamber was used, where male Sprague-Dawley rats fed on either standard or cholesterol enriched (HC) diets, as well as streptozotocin diabetics, were exposed to SO2 at 5 and 10 ppm, 24 h per day for 14 days. In rats, both on a standard diet and on a HC regimen, SO2 exposure determined a significant dose-dependent increase in plasma triglycerides, up to +363% in the 10 ppm HC exposed animals. This same gas concentration significantly reduced HDL cholesterol levels. In contrast, exposure of diabetic animals to 10 ppm SO2 resulted in a fall (−41%) of plasma and liver triglycerides and in a concomitant increase (+62%) of plasma HDL cholesterol. This discrepancy could apparently be related to diverging effects of SO2 exposure on plasma insulin levels in the different animal groups. Kinetic analyses of triglyceride synthesis carried out in rats on a standard diet revealed, in exposed animals, a significant reduction in the secretory rate, in spite of the concomitant hypertriglyceridemia. These findings suggest that SO2 exposure can markedly modify major lipid and glycemic indices, also indicating a differential response in normo/hyperlipidemic versus diabetic animals.

Dietary animal proteins and cholesterol metabolism in rabbits

The effect in rabbits of giving isonitrogenous purified diets containing casein, ovalbumin, fish protein, milk-whey protein and soya-bean protein were compared. The diets were balanced for cholesterol and for the amount and type of fat. When incorporated into low-cholesterol diets (0.08 g cholesterol/kg), casein, ovalbumin and soya-bean protein produced similar levels of serum cholesterol. With a high background of dietary cholesterol (1.5 g/kg), serum cholesterol concentrations increased with soya-bean protein, whey protein, casein and fish protein, in that order. Thus, the hypercholesterolaemic effect of casein in carefully balanced diets was only seen against a high-cholesterol background. The development of hypercholesterolaemia produced by giving fish protein was different from that produced by casein. First, less cholesterol accumulated in the very-low-density-lipoprotein fractions and more in the lipoproteins of higher density with fish protein than with casein. Second, fish protein, unlike casein, did not increase liver cholesterol. Third, transfer of rabbits from a diet containing soya-bean protein to one containing casein resulted in an immediate marked depression in neutral steroid and bile acid excretion in faeces. However, when rabbits were fed on the diet with fish protein after the diet with soya-bean protein, there was no significant depression in neutral steroid output and the depression in bile acid output was delayed. The present study suggests that different animal proteins cause hypercholesterolaemia by different mechanisms.

Increased plasma and aortic triglycerides in rabbits after acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Administration of a single nonlethal dose (20 micrograms/kg) of 2,3,7,8-tetrachlorodibenzo-p-dioxin to New Zealand male rabbits, both on a standard and on a cholesterol (0.5% in the diet) regimen, resulted in a significant increase of plasma triglyceride levels. Triglycerides were particularly raised in the very low-density lipoprotein fraction; no significant apolipoprotein changes, as assessed by an analytical isoelectrofocusing procedure, could be determined. Concomitant to the increased triglyceridemia, aortic triglycerides were also significantly elevated in 2,3,7,8-tetrachlorodibenzo-p-dioxin-pretreated rabbits, both on the standard and on the cholesterolemic regimen. These findings suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin, possibly by inhibiting triglyceride breakdown, may induce an atherogenic form of hypertriglyceridemia in a standard experimental model of atherosclerosis.

Pantethine reduces plasma cholesterol and the severity of arterial lesions in experimental hypercholesterolemic rabbits

Pantethine (P), a coenzyme A precursor, was administered to cholesterol-fed rabbits (0.5% cholesterol diet + 1% pantethine) for 90 days. At the end of treatment, plasma total cholesterol levels were reduced 64.7% and the HDL/total cholesterol ratio increased in P-treated animals; a significant rise of the apo A-I/A-II ratio was detected in HDL. VLDL lipid and protein levels were, on the other hand, reduced by P. The cholesterol-ester content of both liver and aortic tissues was not significantly affected by P. Although the total aortic area with evident plaques was reduced only 18.2%, the microscopical examination of sections from the major vessels of P-treated animals, showed a reduction in the severity of lesions, both in the aorta and in the coronary arteries. These findings suggest that P, in addition to significantly lowering plasma cholesterol levels in rabbits on an experimental diet, may modify lipid deposition in major arteries, possibly by affecting lipoprotein composition and/or exerting an arterial protective effect.

Mechanisms of Lipid-Lowering Agents

Lipid-lowering agents are used with the purpose of ameliorating hyperlipoproteinemias, in order to prevent arterial disease. Lipid-lowering drugs can be classified into absorbable agents and into nonabsorbable compounds, acting within the gastrointestinal lumen. Absorbable drugs (fibric acids, nicotinic acid, probucol, HMG-CoA reductase inhibitors) reduce plasma very-low-density lipoproteins (VLDL) and/or low-density lipoproteins (LDL) by a variety of mechanisms. Fibric acids, in particular, act by stimulating the catabolism of VLDL and also, as a consequence, improving LDL delipidation, thus favoring receptor uptake. Nicotinic acid and acipimox interfere with the biosynthesis of LDL and can also improve the clearance of VLDL/LDL. Probucol acts by a newly described mechanism, i.e. accelerating reverse transport of cholesteryl esters from high-density lipoproteins to lower-density lipoproteins. Finally, HMG-CoA reductase inhibitors, interfering with the biosynthesis of cholesterol, can induce an increased expression of liver high-affinity lipoprotein receptors. Nonabsorbable agents (anion-exchange resins, neomycin, beta-sitosterol) interrupt the recirculation of bile acids and/or reduce the absorption of cholesterol with the gut. They display a selective activity on hypercholesterolemia, again by increasing LDL receptor expression. The choice of one or more lipid-lowering agents will depend upon the patients phenotype, determining responsiveness to the pharmacological treatment.

Soy milk with a high glycitein content does not reduce low-density lipoprotein cholesterolemia in type II hypercholesterolemic patients.

In order to evaluate acceptability and effectiveness of a partial addition of soy protein to the daily diet in well-established type II hypercholesterolemic individuals, a double-blind study was carried out with a soy milk providing 25 g/day of protein versus an identically formulated cow’s milk. Twenty patients with type II hypercholesterolemia, 4 males and 16 females, age range 38–76 years, all with cholesterol levels >7 mmol/l and low-density lipoprotein cholesterol <5.5 mmol/l, were selected. Significant triglyceride elevations (WHO Fredrickson type IIB) were present in 4 patients, and the body mass index was 24.2 ± 3.47 kg/m2. Different from prior studies, either with isoflavone-free products or with a moderately isoflavone-rich milk, the milk in the present study did not reduce total and low-density lipoprotein cholesterolemia. A detailed analysis of the composition showed significant differences in the isoflavone content versus products used in prior studies with a positive outcome. Soy milk isoflavones were, in fact, characterized by a high glycitein content and a low genistein/daidzein ratio. Glycitein is a minor component in most soy products, and its role in cholesterol regulation is unclear. In view of the high interest in the use of soy products for the prevention of coronary diseases, the metabolic behavior of different isoflavones in man should be better characterized, and the role of isoflavone composition of soy products given for the control of cholesterolemia needs further clarification.

Differential effects of beclobrate on lipid/lipoprotein distribution in normal and hypercholesterolemic rats.

Beclobrate, a new fibric acid derivative, displays remarkable lipid lowering activity in rodents. In order to evaluate changes in the distribution and liver handling of lipoproteins, beclobrate was tested in rats fed on a normal or hypercholesterolemic diet. On the normal diet, beclobrate lowered total plasma cholesterol by 22-33.4% (10-50 mg/kg); the cholesterol reduction occurred mainly in high density lipoproteins (HDL) (by 24-45% with the three tested doses). The metabolic clearance of 125I-labelled beta-very low density lipoproteins (beta-VLDL) injected into these animals almost doubled (0.20 1/h vs. 0.13 1/h in controls) after treatment with 20 mg/kg of beclobrate. In addition, beclobrate administration dramatically increased the activity of the high-affinity receptors for beta-VLDL in isolated liver membranes (Bmax: 208 +/- 17.6 vs. 146 +/- 2.6 ng/mg of protein for controls). On the hypercholesterolemic diet, beclobrate treatment (50 mg/kg) was associated with a 25% reduction in total cholesterol accompanied, however, by a 166% rise in HDL cholesterol. In these animals, the composition of VLDL, typically cholesterol-enriched, became close to normal. The increased HDL was characterized by a remarkable enrichment with particles containing apolipoprotein E (apo E), which is compatible with either an improved peripheral cholesterol removal or an enhanced direct secretion of apo E. The two models offer different opportunities for evaluating the mechanism of action of this new lipid lowering agent. Lipoprotein catabolism and receptor-mediated clearance were characteristically improved in normolipidemic rats whereas major effects on HDL metabolism could be demonstrated in hypercholesterolemic rats.

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Effects of pantethine on different models of experimental hyperlipidemia in rodents: A comparison with clofibrate

Summary Pantethine was tested in different forms of experimental hyperlipidemia in a rodent strain (Ivanovas-Sieve) spontaneously exhibiting elevated plasma triglyceride (TG) levels. Pantethine (P) generally given at a daily dose of 500 mg/kg, was compared with clofibrate (200 mg/kg). P and clofibrate (CPIB) proved ineffective on ACTH lipolysis and on Triton hyperlipidemia; CPIB showed a significant activity on fructose and ethanol induced hypertriglyceridemia; both compounds effectively reduced elevated TG and cholesterol levels following the Nath diet. In all experimental models, both in basal conditions and after stimulation, CPIB generally reduced high density lipoprotein cholesterol (HDL-C) levels; this effect was not elicited by P.

Endogenous hypertriglyceridemia in a nonobese rat model: Plasma lipoproteins and dietary sensitivity

Sprague-Dawley male rats from the Ivanovas-Sieve colony (IVA-SIV) show higher plasma triglyceride levels compared to the standard Charles River (CR) rats. The triglyceride enrichment occurs primarily in the very-low-density lipoproteins (VLDL), otherwise of a normal % composition, suggesting that the number of particles is increased, rather than their triglyceride (TG) content. High-density lipoprotein (HDL) particles, corresponding to HDL1, appear to be increased in the IVA-SIV rats, as confirmed by rate-zonal ultracentrifugation. The apoprotein composition of isolated lipoproteins (apo B content and isoelectric focusing pattern), does not differ in the two strains. The IVA-SIV rats are remarkably more TG inducible, compared to the standard CR. This can be shown with fructose loading and with a cholesterol-cholic acid diet. The plasma TG increase, after Triton administration, indicative of the VLDL-TG production, is fourfold higher in the IVA-SIV, compared to the CR rats. These findings provide evidence for some similarities between the IVA-SIV rat and human endogenous hypertriglyceridemia, and suggest an increased TG biosynthesis in this model.