Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids.

Morphological and biochemical effects were induced at the subcellular level in the skeletal muscle, heart and liver of male rats as a result of feeding with EPA, DHA, and 3-thia fatty acids. The 3-thia fatty acid, tetradecylthioacetic acid (TTA) and EPA induced mitochondrial growth in type I muscle fibers in both the diaphragm and soleus muscle, and the size distribution of mitochondrial areas followed a similar pattern. Only the 3-thia fatty acid induced mitochondrial growth in type II muscle fibers. The mean area occupied by the mitochondria and the size distribution of mitochondrial areas in both fiber types were highly similar in DHA-treated and control animals. Only the 3-thia fatty acid increased the gene-expression of carnitine palmitoyltransferase (CPT)-II in the diaphragm. In the heart, however, the gene expression decreased. In hepatocytes an increase in the mean size of mitochondria was observed after EPA treatment, concomitant with an increase in mitochondrial CPT-II gene expression. Administration of 2-methyl-substituted EPA (methyl-EPA) induced a higher rate of growth of mitochondria than EPA. At the peroxisomal level in the hepatocytes a 3-thia fatty acid, EPA, and DHA increased the areal fraction concomitant with the induction of gene expression of peroxisomal fatty acyl-CoA oxidase (FAO). In the diaphragm, mRNA levels of FAO were not affected by EPA or DHA treatment, whereas gene expression was significantly increased after 3-thia fatty acid treatment. In the heart, both 3-thia fatty acid, EPA and DHA tended to decrease the levels of FAO mRNA. The areal fraction of fat droplets in all three tissue types was significantly lower in the groups treated with 3-thia fatty acid. In the group treated with EPA a lower areal fraction of fat droplets was observed, while the DHA group was similar to the control. This indicates that EPA and DHA have different effects on mitochondrial biogenesis.

Midazolam in combination with fentanyl/fluanisone and nitrous oxide as anaesthesia in rabbits--cardiovascular parameters.

When establishing a rabbit model for cardiovascular research in our laboratory we have used midazolam in combination with fentanyl/fluanisone (MFF) and nitrous oxide as anaesthesia. In this study we focused on the effect of the anaesthetic regimen on cardiovascular parameters during open-chest surgery in 12 rabbits. Rabbits were tranquillized by intramuscular injection of fentanyl/fluanisone (0.2 ml/kg of the drug that contained 10 mg/ml fentanyl and 0.2 mg/ml fluanisone). After an intraperitoneal injection of midazolam (4 mg/kg) and additional i.m. injection of fentanyl/fluanisone (0.1 ml/kg) the rabbits were tracheotomized and ventilated on a respirator delivering a gas mixture of 50% N2O, 47.5% O2, and 2.5% CO2. The femoral vein and artery were cannulated and then rabbits received a supply of MFF intravenously. The chest was opened by midline sternotomy and the left ventricle was instrumented with piezo-electric crystals for measurement of regional left ventricular function and with a pressure catheter to measure left ventricular pressure. Radiolabelled microspheres were used to assess cardiac output and left ventricular tissue blood flow. Blood gas analysis showed no difference in the values of pH, pCO2 and pO2 between the open-chest and the closed-chest states. Mean aortic pressure was 74 +/- 4 mmHg in the closed-chest state and 65 +/- 4 mmHg in the open-chest state. Tissue blood flow showed that the left ventricle was well perfused, and mean tissue blood flow values varied between 1.80 and 2.36 ml/min.g. We conclude that the anaesthetic regimen used is easy to control. It is well tolerated in rabbits and is suitable for studies on myocardial contraction in rabbits.

Tetradecylthioacetic acid reduces the amount of lipid droplets, induces megamitochondria formation and increases the fatty acid oxidation in rat heart.

The effects of prolonged administration (3 months) of a 3-thia fatty acid analogue and omega-3-fatty acids on cardiac fatty acid oxidation and the volume fraction of lipid droplets and mitochondria in cardiomyocytes were investigated. Doses were 1 g/day/kg body weight, except 150 mg/day/kg body weight for tetradecylthioacetic acid (a 3-thia fatty acid). One group served as control and did not receive any treatment. The volume fraction of lipid droplets in cardiomyocytes was significantly lower in the tetradecylthioacetic acid group compared to the other groups. Mitochondrial beta-oxidation was 60% greater and fatty acyl-CoA oxidase activity was increased by 430% in the tetradecylthioacetic acid group compared to control. This was accompanied by a greater volume fraction of mitochondria in cardiomyocytes (0.514 +/- 0.032% in tetradecylthioacetic acid v 0.318 +/- 0.007% in control) which was due to an increased size of mitochondria. The volume fraction of mitochondria was also greater in eicosapentaenoic acid (EPA) treated rats compared to control, but the enzymic activities were unaffected. Docosahexaenoic acid (DHA) treatment resulted in a greater volume fraction of lipid droplets in the cardiomyocytes, but the volume fraction of mitochondria and enzyme activities were unaltered. These results indicate that EPA and DHA have different effects on the modulation of mitochondrial biogenesis. Tetradecylthioacetic acid treatment results in megamitochondria formation and increased peroxisomal and mitochondrial beta-oxidation with a concomitant reduction of lipid droplets in the cardiomyocytes.

Effects of endurance training on left ventricular performance: a study in anaesthetized rabbits.

Endurance training is known to increase ventricular performance during exercise and to decrease resting heart rate. The aim of this study was to evaluate a model for endurance training in rabbits and to study the effects of endurance training on local myocardial performance in the left ventricle during resting conditions. One group of rabbits underwent a 10-week exercise training programme. The rabbits trained 5 days a week on a treadmill. Training periods increased gradually from 15 min to 1 h with increments in speed from 0.5 to 1.2 km h-1. After the training programme the rabbits were anaesthetized and studied as acute open-chest preparations. A micro-tip pressure transducer was introduced via apex to the left ventricle and two pairs of ultrasonic crystals were implanted in the left anterior wall to measure segment lengths. One pair measured shortening in the circumferential direction whereas the other pair measured shortening in the longitudinal direction. Heart rate was lower in the trained group (n = 5), 172 +/- 9 beats min-1 (mean +/- SEM), compared with 235 +/- 19 beats min-1 in the control group (n = 8) (P < 0.02). Stroke volume, measured by radio-nuclidelabelled microspheres, was greater in the trained rabbits compared with controls (P < 0.03). Shortening in both segments was of similar magnitude for the trained and control groups. End-systolic pressure-length relations (ESPLR) obtained by occlusion of the descending aorta (balloon catheter) showed reduced slopes for longitudinal segments in the trained group compared with the control group (P < 0.05). We conclude that this endurance training programme in rabbits can be used to study myocardial effects of endurance training. Furthermore, the less steep slope of ESPLRs for the longitudinal segment in the trained animals might indicate a structural myocardial remodelling and increased contractile reserve that might be recruited during adrenergic stimulation in the trained group.

Eicosapentaenoic acid causes transient accumulation of lipids in rat myocardium.

Rats were given eicosapentaenoic acid (EPA) or palmitic acid (PALM) up to 15 days, and control animals were given carboxymethylcellulose. All suspensions which were given by gastric intubation contained tocopherol. Heart triacylglycerols, heart cholesterol and heart phospholipids significantly increased after one day of EPA treatment, but they were normalized within 15 days. Both after 2 and 10 days of treatment with palmitic acid the heart triacylglycerols were significantly greater than control. The heart cholesterol and heart phospholipids were significantly greater than control after 10 days of treatment with palmitic acid. Total carnitine palmitoyltransferase (CPT) activity in heart was significantly greater in rats treated with EPA for 15 days compared to control, but treatment with palmitic acid had no effect. The fatty acyl-CoA oxidase activity was greater in rats treated with EPA for 15 days and palmitic acid for 10 days compared to control. The fractional volume of lipid droplets in myocardial cells was calculated from electronmicrographs and was 0.112 +/- 0.016% after 1 day of EPA treatment compared to 0.035 +/- 0.016% in the control group. After 5 and 15 days the fractional volume was the same as control. The fractional volume of lipid droplets in rats treated with palmitic acid for 10 days was 0.120 +/- 0.023%. Treatment with EPA caused an immediate accumulation of lipids and lipid droplets in the rat heart which after few days normalized in parallel with an increased activity of total CPT in the heart.

Intravenous lipid infusion results in myocardial lipid droplet accumulation combined with reduced myocardial performance in heparinized rabbits.

The question addressed in this study was whether a relation between myocardial lipid droplet accumulation and depressed myocardial function existed following intralipid infusion for 45 min in open chest, anaesthetized rabbits. One group of rabbits (n = 8) received intralipid infusion whereas a control group (n = 8) received sodium chloride. Local myocardial performance was obtained by sonomicrometry and the fractional volume of myocardial lipid droplets was measured by morphometric methods. The fractional volume of lipid droplets was 0.667 +/- 0.116% in the intralipid group compared with 0.318 +/- 0.080% in the control group (P < 0.03). Cardiac output and stroke volume fell 26% (P < 0.0001) and 34% (P < 0.0001), respectively, as a result of intralipid infusion. However, myocardial blood flow obtained by radiolabelled microspheres remained unchanged. Local myocardial function was reduced for both segments after intralipid infusion; maximal systolic shortening was reduced from 15.63 +/- 1.45 to 12.07 +/- 1.55% (P < 0.002) in the circumferential segment and from 9.46 +/- 1.17 to 7.40 +/- 0.53% (P < 0.05) in the longitudinal segment. The end-diastolic length of the circumferential segment was reduced by 3% (P < 0.05) after intralipid infusion. The reduced end-diastolic length of circumferential segments together with unchanged left ventricular end-diastolic pressure might indicate reduced left ventricular end-diastolic compliance. We conclude that acute intralipid infusion in rabbits results in myocardial lipid droplet accumulation and depressed local myocardial function.

Docosahexaenoic acid induces lipid accumulation in myocardial cells of rats.

The aim of the study was to explore whether treatment with highly purified docosahexaenoic acid (DHA) over a short period affects the amount of lipid droplets in myocardial cells of rats, and whether heart peroxisomal enzyme activity is changed. Fifteen rats were fed a standard diet for 10 days and 15 rats were fed a cholesterol diet (2% of cholesterol) for 10 days. In each experiment six rats served as control, and three rats in each treatment group were given one of the following treatments by gastric intubation: DHA at 500, 1000, or 1500 mg day-1 kg-1 body weight. The fractional volume of lipid droplets in myocardial cells was calculated by morphometric methods. The heart triglycerides and the volume fraction of lipid droplets in the myocardium were greater in the standard diet rats treated with DHA compared with controls. There was no such increase caused by DHA treatment in the cholesterol diet rats. The heart fatty acyl-CoA oxidase tended to increase with DHA treatment in both standard and cholesterol diet rats, but this was significantly increased only after treatment with DHA 1500 mg day-1 kg-1 in the cholesterol diet rats. We conclude that treatment with highly purified DHA for 10 days results in cardiac lipidosis, assessed both by biochemical and morphological methods in standard diet rats, whereas DHA treatment has no additive effect on lipid accumulation in cholesterol fed rats.

A study on lipid metabolism in heart and liver of cholesterol- and pectin-fed rats.

Pectin is known as a cholesterol-reducing dietary fibre, and in the present study we addressed the question whether pectin affected the quantity of lipid in droplets in the myocardial cells and of lipid in the liver cells. Male Wistar rats received either a diet containing cholesterol or a standard diet without cholesterol with 0, 50 or 100 g pectin/kg incorporated for 10 d. The fractional volume of lipid droplets in the myocardial cells decreased as a function of pectin dose in both the standard-fed and the cholesterol-fed rats. Serum cholesterol was significantly reduced in both groups after addition of 100 g pectin/kg diet. The cholesterol diet increased the liver cholesterol level, and 100 g pectin/kg diet resulted in a lower concentration of liver cholesterol in the cholesterol-fed animals, but the influence on standard-fed rats was modest. Hydroxymethylglutaryl-CoA reductase (EC 1.1.1.88; HMG-CoA reductase) activity increased when pectin was given in the standard diet. Liver triacylglycerol level increased after cholesterol and pectin feeding. Mitochondrial fatty acid oxidation and phosphatidate phosphohydrolase (EC 3.1.3.4) activity tended to decrease, whereas the peroxisomal fatty acid oxidation and acyl-CoA oxidase activity were unchanged. Increased hepatic triacylglycerol content by cholesterol and pectin treatment may be due to inhibited mitochondrial fatty acid oxidation along with increased availability of fatty acid for esterification and triacylglycerol synthesis. The presence of pectin in the diets of cholesterol-fed rats resulted in increased hepatic concentration of triacylglycerols and increased mitochondrial fatty acid oxidation. In this case the hepatic accumulation of triacylglycerol may be mediated by a reduced efflux of triacylglycerols from the liver.

The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis.

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal beta-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [14C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export, e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.

Cholesterol induced lipid accumulation in myocardial cells of rats.

OBJECTIVE: The aim was to investigate whether cholesterol feeding of rats for only 10 d would result in lipid accumulation in myocardial cells and a change in the peroxisomal beta oxidation in the heart compared to the hearts of rats on standard feeds. METHODS: Eight rats received a cholesterol diet (2%) and eight rats received a standard diet for 10 d. Lipids were measured in serum, liver, and heart. Palmitoyl-CoA hydrolase and fatty acyl-CoA oxidase were determined in total homogenate of hearts. The fractional volume of lipid droplets in myocardial cells was calculated from micrographs at a magnification of x9600 obtained by electron microscopy. RESULTS: The fractional volume of lipid droplets in the cardiomyocytes increased from 0.109(SEM 0.019)% to 0.259(0.037)%, (p < 0.003), as a result of cholesterol feeding. Cholesterol and triglycerides in the heart measured by biochemical methods increased by 13% and 24%, respectively. There was no difference in palmitoyl-CoA hydrolase or fatty acyl-CoA oxidase in the hearts of the cholesterol fed group compared to the control group, suggesting an unaltered peroxisomal beta oxidation of fatty acids in the myocardium. The serum triglycerides and serum phospholipids were reduced in the cholesterol fed rats (p < 0.05 and p < 0.001, respectively). Rats fed cholesterol diet showed a reduced ratio of HDL cholesterol to total cholesterol in serum, together with an increased liver weight and relative liver weight compared to the control rats. The addition of cholesterol to the diet increased liver cholesterol to 21.0(1.6) mumol.g-1 wet weight compared to 5.87(0.93) mumol.g-1 in the controls on standard diet (p < 0.0001). Cholesterol feeding had no effect on the hepatic level of phospholipids, but the liver triglycerides tended to increase from 23.3(9.5) mumol.g-1 wet weight to 53.3(6.3) mumol.g-1. CONCLUSIONS: Lipid accumulation in the myocardial cells was shown by morphometry after 10 d of cholesterol feeding, without any visible damage to the tissue.

Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.

The effect of docosahexaenoic acid (DHA) on mitochondrial and peroxisomal fatty acid oxidation and on key enzymes in triglyceride metabolism was investigated in the liver of rats fed a standard diet, a cholesterol diet, and a pelleted chow diet. Unexpectedly, in all three rat models repeated administration of highly purified DHA (92% pure) at different doses and times, at a dose of 1000 mg/day per kg body weight, resulted in no significant decrease of hepatic and plasma concentration of triglycerides. The serum concentrations of cholesterol and phospholipids showed an increase in a time-dependent manner in rats fed the pelleted chow diet. The hepatic concentration of cholesterol was increased in rats fed the cholesterol diet and pelleted chow diet after administration of DHA compared to palmitic acid. In all rat models, treatment with DHA tended to increase the peroxisomal beta-oxidation. This was accompanied with a significant increase (1.5-fold) of fatty acyl-CoA oxidase activity. The mitochondrial fatty acid oxidation system and carnitine palmitoyl-transferase activity, however, were almost unchanged. Moreover, palmitoyl-CoA synthetase activity was increased, whereas the palmitoyl-CoA hydrolase activity was decreased. Neither microsomal phosphatidate phosphohydrolase activity nor cytosolic phosphatidate phosphohydrolase activity was affected by DHA feeding in the three rat models. Acyl-CoA:1,2-diacylglycerol acyltransferase activity was also unaffected. In contrast to docosahexanoic acid feeding, eicosapentaenoic acid (EPA) administration possessed a hypotriglyceridemic effect and resulted in an increase of mitochondrial and peroxisomal oxidation of fatty acids. Carnitine palmitoyltransferase activity was also stimulated. Phosphatidate phosphohydrolase activity was unaffected whereas diacylglycerol acyltransferase activity was increased by EPA treatment compared with palmitic acid feeding. The results indicate that docosahexaenoic acid, in contrast to eicosapentaenoic acid, does not inhibit the synthesis and secretion of triglycerides in the liver. In addition, the results emphasize the importance that stimulation of peroxisomal beta-oxidation by these n-3 fatty acids is not sufficient to decrease the serum levels of triglycerides. In addition, increased mitochondrial beta-oxidation of fatty acids and thereby decreased availability of nonesterified fatty acids may be a mechanism by which EPA inhibits triglyceride, and subsequently very low density lipoprotein-triglyceride, production. Whether DHA and EPA possess different metabolic properties should be considered.