Fractal and stereological analyses of insulin-induced rat exocrine pancreas remodelling.

BACKGROUND: The effect of insulin on the endocrine pancreas has been the subject of extensive study, but quantitative morphometric investigations of the exocrine pancreas are scarce. This study was therefore undertaken to investigate the effect of acute and chronic insulin administration (two doses, 0.4 IU and 4 IU) on the morphology of rat pancreas acini. MATERIALS AND METHODS: Semi-fine sections stained with methylene blue and basic fuchsine or haematoxylin and eosin-stained 5-micrometer thick paraffin sections were used for fractal and stereological analysis of exocrine acini. Acute insulin treatment, independent of applied doses increased fractal dimension in line with decreased lacunarity of pancreas acini. Chronic low dose insulin decreased fractal dimension and increased lacunarity of pancreas acini, but a high dose had the opposite effect. The volume densities (Vv) of cytoplasm, granules and nucleus are affected differently: acute low dose and high chronic dose significantly decreased granules Vv, and in line increased cytoplasmic Vv, whereas other examined structures showed slight changes without statistical significance. RESULTS: The results obtained from this investigation indicate that insulin treatment induced structural remodelling of the exocrine pancreas suggesting a substantial role of insulin in its functioning. CONCLUSIONS: Additionally, we showed that fine architectural changes in acini could be detected by fractal analysis, suggesting this method as an alternative or addition to routine stereology.

Expression pattern of thermogenesis-related factors in interscapular brown adipose tissue of alloxan-treated rats: beneficial effect of L-arginine.

Metabolic abnormalities underlying diabetes can be abrogated by L-arginine. Here we examined the molecular basis of disturbed interscapular brown adipose tissue (IBAT) thermogenesis and the possible role of nitric oxide (NO) in the IBAT of diabetic rats. To induce diabetes, adult Mill Hill hybrid hooded male rats were given a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were further divided into three subgroups receiving: (i) L-arginine.HCl (2.25%) or (ii) N(omega)-nitro-L-arginine methyl ester (L-NAME.HCl, 0.01%) for 12 days in drinking water and (iii) untreated controls. Treatment of the diabetic animals started after diabetes induction (glucose level 12 mmol/L). Diabetes led to a decrease in the mRNA levels of uncoupling protein 1 (UCP1), peroxisomal proliferator activator receptor gamma (PPARgamma) and endothelial NO synthase (eNOS) as revealed by RT-PCR. The diabetic rats had reduced eNOS and inducible NOS (iNOS) protein contents accompanied by low tissue vascularization, a parameter directly related to tissue thermogenic state. Downregulation of glutathione peroxidase (GSH-Px) and catalase (CAT) transcripts were also observed in diabetes. In contrast, the expression level of PPARgamma coactivator-1alpha (PGC-1alpha) mRNA was elevated. Supplementation with L-arginine not only restored diabetes-induced changes in the expressions of these molecules important for IBAT regulation, but also increased the vascularity. Interestingly, L-NAME induced similar patterns of changes in vascularity and PGC-1alpha mRNA level as did l-arginine. In summary, our results provide insight into the molecular basis underlying diabetes-induced metabolic and functional disturbances in the IBAT and suggest a beneficial role for the L-arginine-NO production pathway.

Erythrophagosomal haemolytic degradative pathway in rat brown adipocytes induced by hyperinsulinaemia: an ultrastructural study.

The phagocytosis and degradation of erythrocytes were studied in brown adipose tissue of experimentally hyperinsulinaemic rats. We found that insulin induces intensive erythrophagocytosis by brown adipocytes and their degradation by haemolytic pathway. Ultrastuctural study revealed that haemolytic degradation of erythrophagosomes was characterized by progressive and uniform decrease of erythrocyte matrix density. At the beginning of the degradative process small, clear vesicles resembling primary lysosomes were visible inside the erythrophagosome. With time, the erythrocyte structure totally disappeared and transformed into a fine, granular material within the erythrophagosomal vacuole. Finally, the erythrocyte membrane detached from the phagosomal and clumped into the vacuolar space forming one or several small myelin-like figures. In conclusion, brown adipocytes are capable of performing intensive erythrophagocytic activity when brown adipose tissue is stimulated and blood flow is enhanced. The molecular basis for favouring a haemolytic instead of more common granular erythrophagosomal degradative pathway remains unknown.

Nitric oxide regulates mitochondrial re-modelling in interscapular brown adipose tissue: ultrastructural and morphometric-stereologic studies.

As a complex, cell-specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of L-arginine-nitric oxide (NO)-producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving L-arginine, a substrate of NO synthases (NOSs), or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub-groups - acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. L-Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold-induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in L-arginine-treated rats. Ultrastructural examinations of L-arginine-treated cold-acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well-ordered cristae in their profiles), which however were inactive in L-arginine-receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, L-NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.

New insights into male (in)fertility: the importance of NO.

Infertility is a global problem that is on the rise, especially during the last decade. Currently, infertility affects approximately 10-15% of the population worldwide. The frequency and origin of different forms of infertility varies. It has been shown that reactive oxygen and nitrogen species (ROS and RNS) are involved in the aetiology of infertility, especially male infertility. Various strategies have been designed to remove or decrease the production of ROS and RNS in spermatozoa, in particular during in vitro fertilization. However, in recent years it has been shown that spermatozoa naturally produce a variety of ROS/RNS, including superoxide anion radical (O2 (⋅-)), hydrogen peroxide and NO. These reactive species, in particular NO, are essential in regulating sperm capacitation and the acrosome reaction, two processes that need to be acquired by sperm in order to achieve fertilization potential. In addition, it has recently been shown that mitochondrial function is positively correlated with human sperm fertilization potential and quality and that NO and NO precursors increase sperm motility by increasing energy production in mitochondria. We will review the new link between sperm NO-driven redox regulation and infertility herein. A special emphasis will be placed on the potential implementation of new redox-active substances that modulate the content of NO in spermatozoa to increase fertility and promote conception.

Diapedesis of thrombocytes from capillary into the intercellular space of interscapular brown adipose tissue and their increase by Ca-Sandoz.

Diapedetic capacity of the rat thrombocytes to leave capillaries of the interscapular brown adipose tissue (IBAT) and infiltrate the interstitium has been observed by conventional electron-microscopy. Thrombocytes that reach IBAT interstitium are morphologically completely different from lumenary ones. The interstitial thrombocyte has a prominet head region (1.51 x 2.12 microns) and very long phylopodium (3.43 microns). Experimental conditions which induced drastic changes in morphology of interstitial thrombocytes were: sucrose overfeeding (10% over 2 days); a 24 hour starving after sucrose overfeeding and Ca-Sandoz drinking (480 mg/L Ca2+ during 2 days). The thrombocytes in the IBAT interstitium can be classified as activated according to: a) pseudopode extension; b) swollen open canalicullar system (OCS); c) endocytosis via coated pits and vesicles; and d) structural changes in alpha granules excreted to the interstitium through OCS. In the IBAT interstitium of 24-hour starved rats after sucrose overfeeding, a thrombocytic layer was observed. It was suggested that thrombocyte adrenalin, stored in dense bodies, was selectively included in the IBAT supply without mediation of the central nervous system.

Erythrophagocytosis by brown adipocytes of rat interscapular tissue.

In this investigation the following phenomena were observed: 1. Rat interscapular brown adipocytes were found to be capable of erythrophagocytosis; 2. Before leaving the capillary lumen, erythrocytes took some material from the blood plasma by endocytosis and passed the endothelial junction carrying endocytotic vacuole. Some erythrocytes were in transit: the so-called "head" was in the process of engulfment by brown adipocytes while the rest of the cell had not left the capillary lumen. Fragmentation of erythrocytes was observed during passage through the endothelial junction as well as in the cytoplasm of adipocytes. 3. In some brown adipocytes erythrocytes retained the same shape as in the capillary, but in many cases they exhibited unusual form. Intracytoplasmic erythrocytes were seen in a semithin sections stained with toluidine blue. 4. Erythrocytes either became cells which phagocytized mitochondria and lipid droplets before their transformation into lipofuscin bodies or they were degraded into ferritin-like particles observed (on unstained sections) in the mitochondrial matrix, intercristal space, on the periphery of lipid droplets and in brown adipocyte cytoplasm.

Insulin-induced iron loading in the rat brown adipose tissue: histochemical and electron-microscopic study.

In the present study we have reported an iron-loading in the rat brown adipose tissue (BAT) after 3-day treatment with insulin (4 IU/kg). Light microscopy showed numerous iron-positive cells (Perls' stain) mainly macrophages and brown adipocytes, while electron-microscopic examination revealed lipofuscin granules and phagosomes as iron-containing components. These results clearly indicate that iron participates in damaging of brown adipocytes.

Gastrin producing G-cells after chronic ethanol and low protein nutrition.

Male Wistar rats, (2 months old), randomly divided according to the diet offered to four groups (C-control; A- alcoholized, PD-protein-deprived, A-PD- alcoholized protein-deprived). In group A and A-PD rats, the number of gastrin producing G-cells was significantly lower. The volume density of G-cells was significantly decreased in alcoholic rats. Fasting serum gastrin level (FSGL) significantly raised due to combined effect of alcohol consumption and protein malnutrition. In group A rats, the profile area of G-cells and their nuclei increased. In PD rats, the profile area of G cells also increased. There were no differences in nucleus/cell ratio due to alcohol ingestion alone, but it decreased significantly in PD and A-PD rats. Pale and lucent types of granules were predominantly seen in G-cells of animals of group A and A-PD. Mean diameter of granules increased in A, PD and A-PD rats. Other endocrine cells (ECL, D, EC) also decreased in number in A rats. Somatostatin producing D-cells decreased significantly in A-PD rats, both in fundic and pyloric mucosa.

Calcium-induced alteration of mitochondrial morphology and mitochondrial-endoplasmic reticulum contacts in rat brown adipocytes.

Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca2+]i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.

Endothelial cell apoptosis in brown adipose tissue of rats induced by hyperinsulinaemia: the possible role of TNF-α.

The aim of the present study was to investigate whether hyperinsulinaemia, which frequently precedes insulin resistance syndrome (obesity, diabetes), induces apoptosis of endothelial cells (ECs) in brown adipose tissue (BAT) and causes BAT atrophy and also, to investigate the possible mechanisms underlying ECs death. In order to induce hyperinsulinaemia, adult male rats of Wistar strain were treated with high dose of insulin (4 U/kg, intraperitonealy) for one or three days. Examinations at ultrastructural level showed apoptotic changes of ECs, allowing us to point out that changes mainly but not exclusively, occur in nuclei. Besides different stages of condensation and alterations of the chromatin, nuclear fragmentation was also observed. Higher number of ECs apoptotic nuclei in the BAT of hyperinsulinaemic rats was also confirmed by propidium iodide staining. Immunohistochemical localization of tumor necrosis factor-alpha (TNF-α) revealed increased expression in ECs of BAT of hyperinsulinaemic animals, indicating its possible role in insulin-induced apoptotic changes. These results suggest that BAT atrophy in hyperinsulinaemia is a result of endothelial and adipocyte apoptosis combined, rather than any of functional components alone.

Morphological characteristics of abdominal adipose tissue in normal-weight and obese women of different metabolic profiles.

BACKGROUND AND AIMS: Morphological changes in adipose tissue reflect functional disorders that correlate with cardiometabolic complications of obesity. The metabolic risks vary among the obese individuals. Furthermore, normal-weight individuals are not necessarily metabolically healthy. Therefore, the aim of this study was to analyze morphological characteristics of the abdominal adipose tissue in normal-weight and obese individuals in regards to metabolic risks. METHODS AND RESULTS: The study group consisted of 30 overweight or obese and 20 normal-weight women undergoing elective surgery. Women of each group were divided into metabolically healthy and metabolically obese, based on the homeostasis model assessment of insulin resistance (HOMA-IR), triglyceride, total-, LDL- and HDL-cholesterol levels. The size and numerical density of adipocytes, as well as volume density of blood vessels in subcutaneous and visceral adipose tissue were compared among subgroups. The results showed hypertrophy of adipocytes of visceral adipose tissue in metabolically obese normal-weight women. At the same time, metabolically healthy obese women had smaller adipocytes in both depots in comparison with "at risk" obese women. The lowest volume density of blood vessels correlated with the largest diameter of adipocytes in "at risk" obese women indicating hypoxic changes in visceral adipose tissue. The observed differences of the adipose tissue morphology did not correlate with considerable phenotypic differences within either the normal-weight or obese women group. CONCLUSION: Changes in adipocyte size, cellular and vascular density of adipose tissue in relation with metabolic disorders, regardless of nutritional level, suggest limited capacity of fat deposition and adipose tissue response to hypoxia.

The role of nitric oxide in remodeling of capillary network in rat interscapular brown adipose tissue after long-term cold acclimation.

Cold exposure has been shown to increase blood flow in interscapular brown adipose tissue (IBAT). The aim of the present study was to evaluate the role of the L-arginine-nitric oxide (*NO) pathway on IBAT capillary network remodeling and its possible correlation with superoxide anion radical (O2(*-)). In the rats that received L-arginine (2.25%) or NG-nitro-L-arginine methyl ester (L-NAME, 0.01%) as a drinking liquid and maintained at room (22+/-1 degrees C) or low (4+/-1 degrees C) temperature for 45 days, IBAT capillaries were analyzed by stereology and observed by light and electron microscopy. Additionally, endothelial *NO synthase (eNOS) expression, nitrotyrosine immunoreactivity and both copper zinc superoxide dismutase (CuZnSOD) enzyme activity and immunohistochemical localization were examined. Stereological analyses of IBAT show that the capillary volume density, as well as capillary-to-brown adipocytes ratio, are increased in cold. L-arginine treatment increases, while L-NAME decreases both parameters, compared to respective controls. Those changes were accompanied by capillary dilatation observed by light and electron microscopy. The activity of CuZnSOD is lower in control cold-acclimated rats, as well as in both L-arginine-treated groups, when compared to control animals acclimated to room temperature. L-NAME treatment attenuates the effects both of cold and L-arginine on CuZnSOD and increases immunopositivity for CuZnSOD in room temperature-acclimated rats. Our results show that *NO induces remodeling of the IBAT capillary network by angiogenesis, and presumably that interaction with O2(*-) has a role in that modulation. The increased eNOS expression accompanied by an increased nitrotyrosine immunoreaction observed in both L-arginine-treated groups compared to corresponding controls strengthens this hypothesis.

Comparative ultrastructural studies on mitochondrial pathology in the liver of AIDS patients: clusters of mitochondria, protuberances, "minimitochondria," vacuoles, and virus-like particles.

The present comparative conventional electron microscopic studies of the liver biopsy in 20 AIDS patients revealed numerous disintegrated mitochondria in hepatocytes, Kupffer cells lymphocytes, and eosinophile leukocytes. It was observed that (1) AIDS mitochondria are clustered, disorganized, and with protuberances; (2) the number of cristae is reduced and they are branched dichotomously; (3) vacuoles are located both within the mitochondria and, in their vicinity, (4) " minimitochondria" are within the ordinary mitochondria; (5) virus-like particles are at the periphery of electron-dense mitochondrial remains.