Fractional Volume Of Lipid Droplets Research Articles

Volumetrijska analiza lipidnih kapi u limfocitima bolesnika sa tip 2 dijabetes melitusom i hiperlipidemijom

Introduction: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by inadequate glucose homeostasis. A common occurrence of T2DM is diabetic dyslipidemia. Given lipid droplets' role in intracellular lipid storage, these structures lie at the center of lipid and energy homeostasis. Lipolysosomes are cell organelles that have the structure of lipid droplets surrounded by a membrane. Lipophagy is a selective form of autophagy that enables lipid droplet degradation, thus representing an important mechanism in the regulation of lipid droplet homeostasis. Aim: The aim of our research was fractional volume analysis of lipid droplets, autophagic vesicles containing lipid droplets, and lipolysosomes in the lymphocytes of patients with T2DM and hyperlipidemia. Material and methods: Mononuclear cells were isolated from the peripheral blood of T2DM patients with hyperlipidemia and from healthy individuals. Cells were fixed in glutaraldehyde and postfixed in 1% osmium tetroxide. After contrasting with 4.7% uranyl acetate, the samples were embedded in epoxy resins and cut by an ultramicrotome. The ultrathin sections were then contrasted with uranyl acetate and lead citrate and analyzed using transmission electron microscopy. The fractional volume of lipid droplets, autophagic vesicles containing lipid droplets, and lipolysosomes was determined using the double "coherent point" grid with dots distributed at two different densities. Results: While there was no difference in the fractional volumes of lipid droplets and autophagic vesicles containing lipid droplets, the fractional volume of lipolysosomes was significantly higher in the lymphocytes of T2DM patients with hyperlipidemia compared to healthy individuals (p < 0.05). Conclusion: A higher fractional volume of lipolysosomes revealed in the lymphocytes of T2DM patients with hyperlipidemia can be due to an increase in the activity of these organelles, as well as an overall increase in cellular lipid metabolism in these patients.

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 carboxymethyl-cellulose. 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 ]0 days of treatment with palmitic acid. Total camitine 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.

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.

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.

Cholesterol induced lipid accumulation in myocardial cells of rats.

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. 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. 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. Lipid accumulation in the myocardial cells was shown by morphometry after 10 d of cholesterol feeding, without any visible damage to the tissue.

Relationship between morphological signs of cell injury in myocardial ischaemia.

Morphological signs of severe ischaemic injury are well known. However, we still lack knowledge about how indices of milder injuries are related on a cellular level. In a previous study we have reported on the sequence of alterations across the border zone in cat hearts subjected to 3 h of coronary occlusion. In the present study, which elaborates that study, we have examined the relationship between morphological variables in serial sections of 220 myocytes within the border zone. In cells with intact sarcolemma and no chromatin changes, the fractional volume of cytoplasm has a bimodal distribution indicating cells with and without oedema. Whereas cells with focal disruptions of the sarcolemma have a moderate oedema, usually localized submembranously, cells with extensive sarcolemmal fragmentation have an extensive oedema. A mild oedema is seen before other signs of severe cell injury, even though more extensive oedema is closely associated with sarcolemmal fragmentation. The fractional volume of mitochondria was smaller, whereas the fractional volume of lipid droplets was larger in cells with oedema than in cells without oedema.

Oxfenicine-induced accumulation of lipid in the rat myocardium

Oxfenicine inhibits myocardial metabolism of nonesterified fatty acids (NEFA). The purpose of the present study was to examine the effects of oxfenicine on triglyceride accumulation and the development of histologically visible lipid droplets. The beta-agonist isoproterenol was used to induce elevated arterial NEFA. Four groups of rats were used in the experiment (12 to 14 rats in each group), and each group received two subcutaneous injections, the second injection 25 min after the first, of oxfenicine-isoproterenol, oxfenicine-saline, saline-isoproterenol and saline twice, respectively. One hour after the second injection, the rats were anesthetized, and the hearts from six rats from each group were quickly removed and frozen for later analysis of triglyceride content. From the remaining rats blood samples were drawn for NEFA analysis, and biopsies were taken from the left ventricular wall before the hearts were frozen in liquid nitrogen and prepared for analysis of esters of carnitine and CoA. Quantitative morphometric techniques were used to determine the fractional volume of lipid droplets in myocardial biopsies. Our results show a marked increase in the triglyceride and lipid droplet content in all groups receiving oxfenicine or isoproterenol. The effect was most pronounced after treatment with both drugs. The close association between the increase in triglyceride and lipid droplet supports the notion that the lipid droplets are composed of triglycerides. Our finding that oxfenicine induces lipid droplet accumulation independent of NEFA increase supports the hypothesis that oxfenicine exerts its effect by inhibiting carnitine acyl transferase.

Lipid accumulation in the myocardium during acute regional ischaemia in cats

Accumulation of lipid material in the myocardium was studied in cat hearts with acute regional ischaemia of 3 h duration. The fractional volume of lipid droplets in cytosol was analysed by electron microscopy of myocardial biopsies using a quantitative stereologic technique. Ischaemic and normally perfused myocardium were identified by fluoresceine injection, and tissue blood flow measurements were performed with labelled microspheres. In normal myocardium only small amounts of lipid droplets were found. A marked accumulation of lipid droplets occurred in borderline tissue between the two types of myocardium, whereas lipid accumulation in ischaemic myocardium was less pronounced. The arterial concentration of nonesterified fatty acids was clearly increased during the 3 h coronary artery occlusion period. Increased triglyceride synthesis from arterial fatty acids, or redistribution of intracellular lipids, are suggested as possible explanations for lipid accumulation during acute myocardial ischaemia.

Propranolol induced lipid accumulation and mitochondrial granularity in myocardial cells.

The ultrastructure of the left ventricular wall was studied in mice given intraperitoneal injections of propranolol (5 mg or 20 mg.kg-1 b.w. twice a day for 48 h with an additional dose 3 h before death), and in saline injected controls. Injections of propranolol increase the number of lipid droplets in areas of the myocardium. Fractional volume of lipid droplets in myocardial cells determined using quantitative stereological techniques, showed an increase of 280% and 544% in the group receiving the lowest and highest doses respectively. Injections of propranolol were also followed by an increased granularity of the mitochondria.

Effect of isoproterenol on lipid accumulation in myocardial cells.

Plasma free fatty acid (FFA) levels and myocardial accumulation of lipid droplets were studied in mice receiving 85 mg isoproterenol (ISP)/kg b.wt. with and without pretreatment with nicotinic acid (NA) 30 min before ISP injection, and in saline-injected controls. The animals were killed 30 min after the ISP injection. Plasma FFA were determined by a radiochemical method. Lipid droplet accumulation was analyzed by quantitative stereologic techniques. Plasma FFA were raised from 505.8 +/- 15.7 to 1,808.8 +/- 87.8 mumol/l after ISP treatment and to 1,108.0 +/- 34.7 mumol/l in the group receiving NA pretreatment. However, NA pretreatment did not bring about a corresponding decrease in the fractional volume of lipid droplets, as both groups showed an increase of five times that of the controls. These results are discussed in terms of the pathogenesis of ISP-induced lipid accumulation.

Stereological analysis of synchronized hepatoma cells as a function of the cell cycle

Synchronized HTC cells were analyzed by morphometry at various times of the cell cycle. A twofold decrease in the number of lysosomes per unit cellular volume was observed after mitosis, while the fractional volume of these particles was more than doubled at the end of the G1 phase and during the S phase. Total lysosomal membrane area was, however, remarkably stable, notwithstanding the large variations in endocytosis occurring during the cell cycle. Clustering of lysosomes was evaluated on a quantitative basis. In mitotic cells, clusters contain on average a minimum of 15 lysosomes. Although less pronounced than at mitosis, clustering persists throughout the interphase. The number of Golgi complexes which can be identified per unit section area increases fivefold during the interphase, with a maximum during the S phase. The fractional volume of lipid droplets displays a large increase from early G1 until the end of the S phase.