Gordana Cvijic

Fasting induced cytoplasmic Fto expression in some neurons of rat hypothalamus.

Fat mass and obesity associated protein (Fto) is a nucleic acid demethylase, with a preference for thymine or uracil, according to the recent structural data. This fact suggests that methylated single-stranded RNA, rather than DNA, may be the primary Fto substrate. Fto is abundantly expressed in all hypothalamic sites governing feeding behavior. Considering that selective modulation of Fto levels in the hypothalamus can influence food intake, we set out to investigate the effect of 48 h fasting on the Fto expression in lateral hypothalamic area, paraventricular, ventromedial and arcuate nucleus, the regulatory centres of energy homeostasis. We have demonstrated that 48 h fasting causes not only an increase in the overall hypothalamic levels of both Fto mRNA and protein, but also alters Fto intracellular distribution. This switch happens in some neurons of paraventricular and ventromedial nucleus, as well as lateral hypothalamic area, resulting in the majority of the enzyme being localized outside the cell nuclei. Interestingly, the change in the Fto intracellular localization was not observed in neurons of arcuate nucleus, suggesting that fasting did not universally affect Fto in all of the hypothalmic sites involved in energy homeostasis regulation. Both Fto mRNA and catechol-O-methyltransferaze mRNA were upregulated in the identical time-dependent manner in fasting animals. This fact, combined with the knowledge of the Fto substrate preference, may provide further insight into monoamine metabolism in the state of disturbed energy homeostasis.

Serotonin-producing enterochromaffin (EC) cells of gastrointestinal mucosa in dexamethasone-treated rats.

The aim of our study was to investigate the morphological, immunohistochemical and ultrastructural changes of rat serotonin-producing enterochromaffin (EC) cells of gastrointestinal mucosa in dexamethasone-treated rats (D). After 12-daily intraperitoneal administration of 2 mg/kg dexamethasone, rats developed diabetes similar to human diabetes type 2. Stomach, small and large intestines were examined. Large serotonin positive EC cells appeared in the corpus mucosa epithelium of D group of rats, although these cells were not present in control (C) rats. Both volume fraction and the number of EC cells per mm(2) of mucosa were significantly increased only in the duodenum. However, the number of EC cells per circular sections of both antrum and small intestine was increased, but reduced both in the ascending and descending colon in D group. The dexamethasone treatment caused a strong reduction in number of granules in the antral EC cells, while it was gradually increased beginning from the jejunum to descending colon. The mean granular content was reduced in the antral EC cells but increased in the jejunal EC cells in D group. In conclusion, the present study showed that morphological changes in gut serotonin-producing EC cells occurred in diabetic rats.

The effect of vasopressin 1b receptor (V1bR) blockade on HPA axis activity in rats exposed to acute heat stress

SUMMARY Thermal stressors such as low and high ambient temperature elicit an abundance of neuroendocrine responses including activation of the hypothalamo-pituitary–adrenal (HPA) axis and arginine vasopressin (AVP) release. The exposure to heat is a particularly interesting model for studying AVP action because this kind of stressor represents not only an unpleasant experience but also a threat to osmotic homeostasis. As AVP has long been recognized as a hormone involved in the modulation of HPA axis activity, the aim of this study was to elucidate the role of AVP in acutely heat-exposed rats using Nelivaptan, a selective vasopressin 1b receptor (V1bR) antagonist. Rats were exposed to high ambient temperature (38°C) for 60 min. The circulating hormones were determined by ELISA or chemiluminescence, and intrapituitary adrenocorticotropic hormone (ACTH) and V1bR level were determined by western blot. The results obtained show that V1bR blockade negatively affected the increase in blood ACTH caused by heat exposure. This treatment alone, or in combination with Nelivaptan, decreased intrapituitary V1bR levels while circulating AVP concentration was increased under the same conditions. Furthermore, a strong correlation was observed between blood ACTH and corticosterone concentration. In conclusion, our results directly confirm the positive role of AVP in the regulation of ACTH secretion from the pituitary in animals exposed to heat. Moreover, the results suggest that AVP from the general circulation influences pituitary V1bR.

The effect of acute heat exposure on rat pituitary corticotroph activation: the role of vasopressin.

The increased ambient temperature affects the function of hypothalamic-pituitary-adrenal (HPA) axis. Since the correlation among vasopressin (VP), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses to various stressors have been long recognized, the aim of this study was to reveal the aforementioned hormones production and morphology of the pituitary gland after exposure to acute heat. Rats were exposed to high ambient temperature (38 °C) for 20 or 60 minutes. The circulating hormones were determined by an ELISA test or chemiluminescences method. The results obtained show the elevation in ACTH and CORT secretion depending on the duration of heat exposure. The VP concentration increased only after prolonged exposure to heat (60 min). The pituitary morphology was examined by routine and fluorescent immunohistochemistry as well as electron microscopy. Observed changes in the anterior and posterior pituitary well corresponded to circulating hormones, regarding the volume density of ACTH-immunopositive cells, percentage of ACTH immunopositive area v. total area and number of VP-immunopositive containing varicose fibers per total area. Acute heat exposure also induced changes in shapes of ACTH-immunopositive cells. Cells appeared stellate with numerous slender cytoplasmic processes and degranulated, which is the most obvious after 20 min. In addition, immunopositivity of endothelial and anterior pituitary cells for VP suggests its influence on ACTH secretion.

Activities of Antioxidant Enzymes and Monoamine Oxidase-A in the Rat Interscapular Brown Adipose Tissue: Effects of Insulin and 6-Hydroxydopamine

The effects of different doses of insulin (INS) (0.4 or 4.0 IU/kg body mass, i.p., for 3 hr) and 6-hydroxydopamine (6-HDA) (100 mg/kg., i.p.) on the activities of antioxidant enzymes--copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT) and catecholamine degrading enzyme monoamine oxidase (MAO-A)--in the rat interscapular brown adipose tissue (IBAT) were studied. In vivo 6-HDA administration, which induces the destruction of sympathetic nerves, markedly reduced IBAT CuZnSOD activity but did not change MnSOD and CAT activities. However, the low dose of INS, which did not induce hypoglycemia, significantly increased the activity of both IBAT mitochondrial enzymes (MnSOD and MAO-A) of control rats. This INS effect on MnSOD was abolished by 6-HDA. On the contrary, CuZnSOD activity was markedly reduced under the influence of INS in both control and 6-HDA-treated rats, whereas for the maintenance of the control level of this enzyme activity, the intact sympathetic nervous system (SNS) is necessary. INS, independent of the dose applied, did not affect CAT activity in control rats, whereas only low INS dose increased the activity of this enzyme in 6-HDA-treated rats. The results indicate that the stimulatory effect of INS on the IBAT mitochondrial enzymes studied is dose dependent and in the case of MnSOD is mediated by SNS. However, the depression in the activity of CuZnSOD is independent of the above-mentioned factors.

Chronic Effect of Insulin on Monoamine Oxidase and Antioxidant Enzyme Activities in the Rat Brainstem

It was shown that hydrogen peroxide (H2O2) is a possible intracellular second messenger in specific insulin action. Because its concentration in the cell depends on the activity of both antioxidant enzymes and monoamine oxidase (MAO), we studied the influence of different insulin doses (0.4 and 4.0 IU/kg body mass, i.p., daily injected over 3 days) on the activity of MAO, types A and B, copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase in the rat brainstem. Chronic insulin treatment significantly increased Vmax of MAO-A and B activities (P < 0.05, P < 0.025, respectively) independent of the dose applied. CuZnSOD activity was also increased (P < 0.025), but only when higher dose of hormone was injected. However, insulin had the opposite effect on MnSOD and catalase causing a decrease in their activities (P < 0.005). The observed changes in the activities of the enzymes studied are possible compensations that potentially maintain an optimal H2O2 level in the brainstem, which might be important for insulin action.

The Activities of Antioxidant Enzymes and Monoamine Oxidase and Uncoupling Protein 1 Content in Brown Fat of Hypo‐ and Hyperthyroid Rats

Abstract: We have studied the activities of antioxidant enzymes (AOE), namely, copper‐zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase (CAT), and the activity of catecholamine‐degrading enzyme monoamine oxidase (MAO) and uncoupling protein 1 (UCP1) content in brown fat (BF) of hypo‐ and hyperthyroid rats. We found that hypothyroidism decreased BF UCP1 content and increased MAO, MnSOD, and CAT activities. T3 increased UCP1 content and MnSOD activity and decreased CuZnSOD, MAO, and CAT activities, while T4 significantly altered (decreased) only CAT activity. This study shows that UCP1 content and MAO and AOE activities in rat BF are notably affected by changed thyroid status.

Effect of acute heat stress on rat adrenal medulla — a morphological and ultrastructural study

Isolated rat adrenal medulla was analyzed by light and electron microscope after an acute (60 min) exposure to high ambient temperature (38°C). Under these conditions there was a significant rise in plasma adrenaline and noradrenaline. Stereological investigation by light microscopy showed a significant decrease in volume density of cells and an increase in the interstitium. At the ultrastructural level, the profile area of cells, nuclei and cytoplasm of adrenaline cells were significantly decreased. After the heat stress numbers of resting granules in adre naline and noradrenaline cells were significantly reduced, while the numbers of altered granules and empty containers in both types of adrenomedullar cells were significantly increased.

Effect of Cold Exposure on Serum DBH and Interscapular Brown Adipose Tissue MAO Activity in Hypothyroid T3- and T4-Treated Rats

Abstract: As the indicators of sympathetic nervous system (SNS) function, the activity of serum dopamine‐β‐hydroxylase (DBH) and interscapular brown adipose tissue (IBAT) monoamine oxidase (MAO) were examined in rats that were chemically thyroidectomized (TX), treated with thyroid hormones, and exposed to cold (4°C). In TX animals, body temperature (bt) significantly decreased, and relative IBAT mass increased as compared with control, euthyroid animals, independent of the ambient temperature. The bt fall in TX cold‐exposed animals was more severe, provoking hypothermia after 4 h. Under the same experimental conditions, the SNS function was enhanced as judged by the increased serum DBH and IBAT MAO activities. The treatment of TX animals with T4 and T3 re‐established the temperature (bt was at the level of controls) and sympathetic homeostasis (DBH activity was at the level of controls) in animals maintained at room temperature but not in those kept under cold conditions. T4 and T3 did not affect IBAT MAO activity of TX rats: It remained significantly above the control values whether the animals were maintained at room temperature or exposed to cold. In conclusion, the IBAT of TX cold‐exposed rats is incapable of responding to the enhanced thermogenic needs despite the increased SNS activity and thyroid hormone substitution.

Cholecystokinin-producing (I) cells of intestinal mucosa in dexamethasone-treated rats.

The aim of this study was to investigate the morphological, immunohistochemical and ultrastructural changes of cholecystokinin-producing (I) cells of gastrointestinal mucosa in dexamethasone-treated rats (D). After 12-daily intraperitoneal administration of 2mg/kg dexamethasone, rats developed diabetes similar to human diabetes mellitus type 2. The mean diameter of the duodenum was significantly decreased due to significant reduction of volume fraction and profile area of lamina propria. There was a decrease in volume fraction and number of cholecystokinin (CCK)-producing cells per mm(2) of mucosa, as well as their numerical density, but without statistical significance. Also, dexamethasone induced appearance of hyperactive duodenal I-cells with small number of granules and dilated endoplasmic reticulum. In conclusion, the present study showed that morphological changes in duodenum cholecystokinin-producing (I) cells occurred in diabetic rats, in a manner which, suggests compensatory effort of CCK cells in diabetic condition.