Volume Density Of Lipid Droplets Research Articles

MORPHOFUNCTIONAL CHANGES IN THE ADRENAL GLANDS IN THE EARLY DEADLINE OF THE DEVELOPMENT OF STREPTOSOTOCIN DIABETES MELLITUS

Relevance of research. The medical and social problem of diabetes involves a steady increase in the prevalence of diseases, high rates of mortality and disability in patients. Disorders of the pituitary-adrenal system play a key role in the pathogenesis of this disease and its complications. Thus, the aim of the research was to determine the specific features of the morpho-functional changes of the adrenal gland in experimental streptozotocin-induced diabetes mellitus and in response to its management. Aim of the study. To establish the peculiarities of morphofunctional restructuring of the adrenal cortex in the early stages (7-14 days) of experimental diabetes. On the 7th day of the development of streptozotocin diabetes mellitus in the adrenal glands, the stage of reactive changes in response to metabolic disorders in the body is traced. Significant thickening of zona fasciculata by 23.95 % (р=0.0007) is noticed in adrenal cortex in this observation period, while the thickness of zona glomerulosa and zona reticularis does not significantly differ from the control indices (р<0.05 in all cases). The endocrinocytes’ profile area is significantly decreased in zona fasciculata that under permanent nucleus area leads to the increase of nucleocytoplasmic index. Morphometric changes in cells are associated with the decrease of lipid droplets in their cytoplasm. It is confirmed by statistical study on the ultrastructural level (volume density of lipid droplets decreases by 48.85 %). These changes in endocrinocytes of zona fasciculata point to significant release of glucocorticoid hormones into blood and is confirmed by biochemical findings, namely, the cortisol level increases by 1.5 times (р=0.0074). Linear proportional associations are revealed between the glucose and cortisol levels r=+0.82 (р=0.0244). On the 14th day of streptozotocin diabetes mellitus is observed to the increase of morphofunctional activity of adrenal cortex, which is evident in: significant thickening of all its zones; significant increase of endocrinocytes’ profile area of all zones and their nuclei; increase of volume density of lipid granules in endocrinocytes of zona fasciculata by 4.73 times respectfully. Blood cortisol level also increases to 18.27±2.21 ng/ml on the 14th day of experiment by 1.7 times.

Seasonal and starvation-induced changes on gonads and lipid reserves of the digestive gland of Nucella lapillus (Caenogastropoda)

This study was conceived to evaluate seasonal changes in the lipid reserves of the digestive gland of the carnivorous intertidal caenogastropod Nucella lapillus, using a stereological approach. Volume density of lipid droplets in the digestive gland, digestive gland weight and volume were assessed in animals collected in March, June, September and December on the Portuguese coast. Gonad development was evaluated to detect any relationship between lipid content in the digestive gland and the reproductive cycle. The quantitative light microscopic analysis demonstrates that lipid droplets are a major component of the digestive gland. In males, the digestive gland and its lipid reserves were quite stable without significant variations throughout the year. In females, the percentage of digestive gland volume occupied by lipid droplets was higher in June and December, coinciding with the highest values of digestive gland volume. Due to the conjugation of these two factors, in June and December the total amount of lipids in the digestive gland was substantially increased in females. In both males and females a relationship between the development status of the gonad and the lipid reserves of the digestive gland was not evident. However, significant differences in the digestive gland lipid reserves were detected between males and females in June and December, pointing to a sex related effect on lipid reserves. To evaluate the use of lipid reserves as energy source in N. lapillus, the consumption of digestive gland lipids was followed during a starvation experiment.

Lipid Content of Non‐cultured and Cultured Pig Embryo

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos. The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage. Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume - V(e), volume density of cytoplasm per unit volume of embryo - Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm - Vv(fat,c) and total volume of lipid droplets per whole embryo - V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).

91 A NOVEL TECHNIQUE FOR EVALUATION OF THE LIPID CONTENT IN PIG EMBRYOS

A high level of lipids, mainly triglycerides and fatty acids, present in embryo cells in the form of lipid droplets is the major factor associated with low cryopreservation of porcine embryos. Previous results demonstrated that the low tolerance of pig embryos to cryopreservation can be increased through reduction of lipid droplet contents. Therefore, in order to improve cryopreservation techniques of porcine embryos, it is fundamental to establish proper culture conditions which ultimately will enable a decrease in lipid content. Unfortunately, there are no precise and efficient methods to evaluate the lipid contents of single pig embryos. Previously used stereological analysis combined with physical serial sectioning (Romek et al. 2007 Reprod. Domest. Anim. in press) is time-consuming, and measurement of triglyceride levels based on enzymatic hydrolysis eliminates other types of lipids from the analysis. Taking the above problems into account, we have developed a new method for evaluation of total lipid content in pig embryos. It is based on visualization of lipid droplets using the specific fluorescent dye Nile red and applying confocal scanning microscopy followed by the Cavalieri method. This method enables measurement of several stereological parameters, especially the volume density of lipid droplets per unit volume of cytoplasm Vv(fat,c), which quantifies most precisely the amount of intracellular lipid. The experiment was carried out on 2- to 4-cell and 8- to 16-cell pig embryos, morulae, blastocysts, and late blastocysts cultured in vitro. Embryos were developed from in vivo-produced zygotes to appropriate stages of development in North Carolina State University (NCSU) 23 medium. For each stage, ten of the embryos were examined. Embryos were denuded and fixed with 2% glutaraldehyde and 2% formaldehyde, stained with 100 nm Nile red (Molecular Probes, Leiden, The Netherlands), and analyzed by means of a confocal microscope LSM 510 Meta (Carl Zeiss MicroImaging GmbH, G�ttingen, Germany). Serial optical sections of each individual embryo were measured by the point counting method, and then the Cavalieri method was used to estimate Vv(fat,). Vv(fat,c) values calculated for embryos at different stages of development were compared by one-way analysis of variance and Tukey's intervals. For cultured pig embryos, volume density of lipid droplets Vv(fat,c) significantly decreased during cleavage from 0.55 µm3 µm–3 at the 2- to 4-cell-embryo stage to 0.46 µm3 µm–3 at the blastocyst stage. The differences between lipid droplet volumes calculated for morulae, blastocysts, and late blastocysts were statistically significant. In conclusion, our new method is more precise, efficient, and quick in comparison to previously used ones. Moreover, we confirmed that the content of total lipids in cultured pig embryo is reduced during its development. This research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26) and Net of Reproduction Biotechnology.

Age-related effects on the zona glomerulosa cells of the rat adrenal cortex

Zona glomerulosa cells of the rat adrenal cortex were studied at 2, 6, 12, 18 and 24 months. Adrenals from unstimulated rats were processed for light and electron microscopy and trunk blood was obtained for the assay of plasma renin activity and aldosterone. Similar assays were made in rats stimulated with adrenocorticotropic hormone (ACTH), 30 min before sacrifice. At light microscopy, no major structural changes were observed; the ultrastructure of most organelles was well preserved and the presence of lipofuscin granules in older animals was remarkable. Acid phosphatase activity was observed in lysosomes and some lipofuscin granules. The quantitative study showed an age-related decrease in cell and nuclear volume (p < 0.01 and p < 0.001 respectively) and an increase in volume density of lipid droplets, lipofuscin granules (p < 0.001), lysosomes and Golgi complex; a decrease in volume density of mitochondria and smooth endoplasmic reticulum (p = 0.052) was also observed. Plasma renin activity and aldosterone level were both reduced (p < 0.001). In stimulated animals, an enhanced aldosterone level was obtained which was higher at 12 months and decreased thereafter. These results show an age-related decrease of aldosterone secretion, consistent with structural and ultrastructural data and related to plasma renin decrement. They also show a decline in the response to aldosterone stimulation by ACTH, indicating a lesser functional ability of zona glomerulosa cells in old animals.

Microvascular response to ischemia, and tissue structure, in normal and atrophied skeletal muscle

The objective of this study was to explain why the normally observed reactive hyperemia in frog sartorius muscle following ischemia is absent when this muscle atrophies. Two possibilities were addressed: (1) absence is due to lowered O 2 consumption, making the muscle more tolerant to ischemia, and (2) absence is linked to impaired vascular function in atrophy. We used 10 frogs after 2–3 months and 8 frogs after 7–14 months of laboratory captivity. Animals in the latter group had a significantly lower sartorius muscle weight, i.e., 85 ± 33 vs 24 ± 11 SD mg. Using intravital video microscopy, we measured red cell velocity in capillaries at the muscle surface, and densities of capillaries with moving (NC PER) and stationary red cells (NC STAT) before and after 30 min ischemia. Ischemia induced a significant temporary increase in overall velocity (from 0.10 to 0.27 mm/sec) in normal muscles, but no increase in atrophied muscles. It resulted in no difference in NC PER between the two groups (preischemic levels in both groups: 15.0 cap/mm of test line), but in a significant difference in NC STAT (3.8 vs 11.5 cap/mm in atrophy). Using light and electron microscopy, we also measured structural and ultrastructural parameters in both groups. In atrophied muscles the mean fiber cross-sectional area was lower (568 vs 1935 μm 2) and anatomical capillary density higher (892 vs 282 cap/mm 2) than in normal muscles. Mitochondrial volume density was not statistically different from the 1.5% level in the normal muscle, while the lipid droplet volume density was larger (2.33 vs 0.58%). The percentage of capillaries with damaged endothelium was larger (33.5 vs 12.6%). Using histology, the white cell volume density per capillary volume was also found to be larger in atrophy (1.96 vs 0.83%). From the discrepancy between the lack of intergroup difference in preischemic NC PER and the 3.2-fold difference in anatomical capillary density we estimate that about 60% of capillaries were perfused with red cells in atrophied muscles. Although the preischemic rate of perfusion in these capillaries was comparable between the two groups, the postischemic response was not: reactive hyperemia was absent in atrophy. Our mitochondrial and lipid volume density data do not support the possibility that this absence was due to lowered O 2 consumption, as these densities did not decrease with atrophy. Recent literature reports show a close relationship between (i) impaired vascular smooth muscle function and loss of endothelial integrity, and (ii) white cell trapping in capillaries and poor postischemic flow. On the basis of these reports and on our own observation that endothelial damage, incidence of white cells in capillaries and postischemic NC STAT increased in atrophy, we propose that the lack of response could reflect an impaired vascular function (both at the arteriolar and at the capillary level) due to deterioration of endothelium during atrophy.

Quantitative electron-microscopic analysis of the in vivo effects of tetrahydrocannabinol on rat preovulatory follicles.

The ultrastructural composition of 4 regions of rat preovulatory follicles was examined using stereological techniques following an intraperitoneal injection of tetrahydrocannabinol (THC), once each day, for 3 days prior to sacrifice on proestrus. Control rats were injected with solvent alone. Changes in nuclear, mitochondrial, lipid droplet and dense-body volume density were measured. No differences in volume density were noted in the theca or antral region of the membrana granulosa. Compared to controls, the mitochondrial volume density decreased in the peripheral region and the corona radiata. The volume density of lipid droplets decreased in the peripheral region. These changes, and their relationship to the roles of these regions in follicular steroidogenesis, are discussed.

Relationship between mitochondrial volume density and capillarity in hamster muscles.

Mitochondrial volume density and lipid droplet-volume density were stereologically determined from electron micrographs of muscle fibers from three hamster muscles: retractor, sartorius, and soleus. The number of capillaries around a fiber, length of capillary-fiber contact, and muscle fiber area were also measured. Glycolytic fibers of the retractor and sartorius had larger cross-sectional areas, lower mitochondrial-volume densities, fewer subsarcolemmal aggregates of mitochondria, and lower capillary-fiber contact length in comparison to oxidative fibers of the retractor and soleus. Values for mitochondrial volume density in the different muscles correlated well (r = 0.97) with resting O2 consumption. The mitochondrial volume densities for each muscle correlated well (r = 0.99) with O2 diffusion coefficients of these muscles. Our results indicate that an analysis of the adequacy of O2 supply to an individual muscle fiber must take into account an interplay among fiber size, percent of the muscle fiber perimeter in contact with a capillary (capillary-fiber contact), and O2 demand of the fiber estimated by mitochondrial volume density.

Morphometric studies on lipoprotein particles in developing rat liver and their corticosteroid-induced changes during the late gestational period.

Changes in lipoprotein particles in hepatocytes of the fetal rat liver have been studied morphometrically from days 15-21 of gestation. On all these days, distinct lipoprotein particles are found within the cisternae of the RER, Golgi complexes and Golgi-derived secretory vesicles. Their mean diameter is 30-31 nm. The number of Golgi complexes per hepatocyte, the lipoprotein particle number per Golgi complex and the volume density of the latter remain unchanged within the developmental period examined. The volume density of lipid droplets, however, shows a significant decrease during this time. Following corticosteroid treatment, the mean diameter of lipoprotein particles, the number of lipoprotein particles per Golgi complex, the volume density of the Golgi complex, and that of the lipid droplets increase significantly within the examined period, whereas the number of Golgi complexes per hepatocyte is reduced. These data support the view that triglyceride production in the fetal liver is directly or indirectly stimulated by corticosteroids administered to the pregnant rat, thus giving rise to larger amounts of hepatic lipoproteins and lipids.

Muscle fibre type characteristics in endurance trained and untrained individuals.

The main fibre types of M. vastus lateralis of 10 trained or untrained male individuals (25-35 years) were quantitatively determined by morphological techniques; the fibre types being defined according to the M-band appearance. The volume density of mitochondria ( Vmit ) was higher in endurance-trained muscles. Vmit was higher in Type 1 than in Type 2 fibres, there being no difference between subtypes of Type 2 fibres. The volume density of lipid droplets ( Vli ) showed a wide range of values both with respect to degree of training and between fibre types. Z-band width was not influenced by endurance training, but was considerably larger in Type 1 than in Type 2 fibres. Discriminant analysis showed that 46% of the fibres, preclassified according to the M-band appearance, would have been correctly allocated on basis of the Vmit . The corresponding value for lipid droplets was 42% and for the Z-band width, 62%. It is concluded that Vmit is not a satisfactory criterion for discriminating between fibre types, especially between Type 2A and Type 2B in trained subjects. The study also shows that endurance training reduces the relative importance of individual-dependent factors in comparison with muscle fibre properties when concerning Vmit .

Alcoholic liver injury in baboons: Transformation of lipocytes to transitional cells

Ultrastructure of the lipocytes (the main perisinusoidal cells) and their alterations in the progression of hepatic fibrosis were studied in liver biopsy specimens of baboons pair-fed with diets containing alcohol, or isocaloric carbohydrate, for up to 112 mo. In control baboons, 97% of the cells in the Disse space were lipocytes characterized by a volume density of lipid droplets occupying > 20% of the cell volume and by the presence of microfilament bundles with associated dense bodies and pinocytic vesicles. Intercellular junctions of the adherens type were present between lipocytes and hepatocytes. After alcohol consumption, the number of lipocytes (as assessed by light microscopy) was significantly decreased in fatty livers and at various stages of hepatic fibrosis; this was associated with a decreased hepatic vitamin A content. In baboons fed alcohol, only 48% of cells were lipocytes, whereas 52% were transitional cells defined by a volume of lipid droplets < 20% of the cell. Like the lipocytes, transitional cells exhibited microfilament bundles, dense bodies, and pinocytic vesicles, and were attached to the hepatocytes by cell junctions. The rough endoplasmic reticulum in transitional cells was conspicuous and had an area significantly greater than that in lipocytes of controls and alcohol-fed animals (69% and 37%, respectively). There was a significant correlation between the percentage of transitional cells as well as the area of their rough endoplasmic reticulum and the degree of hepatic fibrosis. Thus, in baboons fed alcohol, the progression of hepatic fibrosis is associated with transformation of lipocytes to transitional cells characterized by a depletion of lipid droplets and a hypertrophy of the rough endoplasmic reticulum; these transitional cells may play a role in promoting hepatic fibrosis in alcoholic liver injury.

Effects of dietary carnitine on myocardial palmitate oxidation in the aging rat.

Aging is associated with decreases in myocardial fatty acid oxidation and carnitine concentration. The purpose of this study was to investigate the effects of dietary carnitine manipulation on myocardial palmitate oxidation and carnitine content in young adult and middle-aged rats. Rats were fed either a carnitine-free or a carnitine-supplemented diet for nine weeks and killed at ages 6.5 and 18 months. Myocardial carnitine content was unaffected by age or diet. However, in 18 months rats fed a carnitine-free diet, myocardial palmitate oxidation was 77% higher, carnitine palmitoyltransferase activity 39% higher, and lipid droplet volume density 55% higher compared to 18 month rats fed a carnitine-supplemented diet. In 6.5 month rats, dietary carnitine had no effect on these variables. These results indicate that dietary carnitine restriction increases myocardial fatty acid metabolism in middle-aged but not in young adult rats.

Ultrastructural changes in the rat exocrine pancreas after jejunoileal bypass.

The influence of a 90% jejunoileal bypass on the rat exocrine pancreas was studied by morphometrical procedures. In sham-operated animals exocrine acinar cells accounted for 80.3% of the pancreas volume. These cells are composed of 9.9% nuclei, 8.4% mitochondria, 12.2% zymogen granules, 0.3% lipid droplets and 69.2% of a compartment ("ERGLS") composed of endoplasmic reticulum, ribosomes, Golgi areas, lysosomes and the cytoplasmic ground substance. Intestinal bypass did not change the volume density of exocrine cells nor that of nuclei in the cells during the three postoperative months. The means nuclear diameter was approximately the same in both groups. However, the volume density of secretory granules diminished by 50%. This was accompanied by a decrease in mean granular diameter, but not in their numerical density. The volume density of lipid droplets increased 10 fold, that of mitochondria increased slightly from the 15th postoperative day but significantly from the 45th day. The remaining cellular compartment composed of "ERGLS" was not modified by intestinal bypass. These findings suggest that a 90% jejunoileal bypass induces major changes in the composition of pancreatic acinar cells but not in their size.

A stereological and biochemical study of the human liver in uncomplicated cholelithiasis.

A quantitative analysis of needle liver biopsies of patients with untreated, uncomplicated cholelithiasis has been performed to better evaluate eventual changes occurring under medical therapy of gallstones. With the light microscope, the intensity of parenchymal steatosis was variable among the patients but the mean volume density of lipid droplets was significantly increased as compared to normal subjects. In addition, there was a significant increase in the volume density of lipocytes (fat-storing cells) without an increase in the number per square area, suggesting a hypertrophy of individual cells. The data obtained by ultrastructural morphometry show a significant increase in the surface density of the rough endoplasmic reticulum in the hepatocytes, a significant increase of the mitochondrial volume density together with the presence of curled mitochondrial cristae and a slight, inconstant intracellular and intracanalicular bile retention. No qualitative or quantitative changes were observed at the level of the smooth endoplasmic reticulum. The activity of the microsomal enzyme NADPH-cytochrome c reductase as evaluated on liver biopsy material was in the normal range. Some of the features observed in this group of patients are reminiscent of alterations previously described in human or experimental cholestasis and suggest that they might depend on a common underlying disturbance in cholesterol and bile salt metabolism.

Fasting and refeeding in the lactating dairy cow: 2. The recovery of liver cell structure and function following a six-day fast

Cows in early lactation were fasted for 6 days and then refed a normal diet for 70 days. Liver cell structure was monitored throughout the experiment by stereological analysis of needle biopsy samples and liver cell function was assessed by the activities of aspartate aminotransferase, sorbitol dehydrogenase and concentrations of bilirubin in the serum. The main changes in the liver of the fasted cows were a decrease in volume density of cytoplasm occupied by glycogen, and an increase in volume density of lysosomes and lipid, both lipid droplets and liposomes. There was a decrease in the number of mitochondria per cell with an increase in mean mitochondrial volume. The changes in liver structure were accompanied by a rise in serum enzyme activity and in the concentration of bilirubin in serum. Liver cell volume and volume density of cytoplasm occupied by glycogen, lysosomes and liposomes returned to control levels within 8 days after refeeding whereas volume density of lipid droplets and the mitochondrial parameters returned to control levels within 16 days. During refeeding there was a close correlation between recovery of liver cell function assessed by blood enzyme concentrations and liver cell structure assessed by stereological techniques.

Morphometric studies on the lipid droplets of the interstitial cells of the renal medulla in different states of diuresis

The volume density of lipid droplets in renal medullary interstitial cells was determined in rats in different diuretic states by electron microscope morphometry. Three different groups of animals were examined: Group I, dehydrated rats deprived of drinking water for 24 hr; group II, acutely hydrated rats treated as group I but given one dose of water 1 hr before sacrifice; group III, water-loaded animals given repeated doses of water prior to sacrifice. In the water-loaded rats which had a low urine osmolality the volume density of interstitial cell lipid droplets was 2.4 times higher than in the dehydrated ones. Correspondingly, the number of lipid droplets was higher by about 46% and the mean single lipid droplet volume was about 64% larger. The volume density of lipid droplets was not significantly different between dehydrated and acutely hydrated animals. Furthermore, there was no difference in urine osmolality between these two groups, suggesting that the acutely hydrated rats were still in a state of antidiuresis. An earlier report of an increased number of lipid droplets in the acutely hydrated rats as compared to the dehydrated ones thus could not be confirmed. It is suggested that the lipid droplets are not secreted from the interstitial cells by exocytosis but are partially or completely removed by intracellular enzymes located in the cytoplasmic ground substance or in cytomembranes.

Acute effects of ACTH on dissociated adrenocortical cells: Quantitative changes in mitochondria and lipid droplets

To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.