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The main pathological hallmark of diabetes is the loss of functional β-cells. Among several types of β-cell death in diabetes, the involvement of ferroptosis remains elusive. Therefore, we investigated the potential of diabetes-mimicking factors: high glucose (HG), proinflammatory cytokines, hydrogen peroxide (H2O2), or diabetogenic agent streptozotocin (STZ) to induce ferroptosis of β-cells in vitro. Furthermore, we tested the contribution of ferroptosis to injury of pancreatic islets in an STZ-induced in vivo diabetic model. All in vitro treatments increased loss of Rin-5F cells along with the accumulation of reactive oxygen species, lipid peroxides and iron, inactivation of NF-E2-related factor 2 (Nrf2), and decrease in glutathione peroxidase 4 expression and mitochondrial membrane potential (MMP). Ferrostatin 1 (Fer-1), ferroptosis inhibitor, diminished the above-stated effects and rescued cells from death in case of HG, STZ, and H2O2 treatments, while failed to increase MMP and to attenuate cell death after the cytokines’ treatment. Moreover, Fer-1 protected pancreatic islets from STZ-induced injury in diabetic in vivo model, since it decreased infiltration of macrophages and accumulation of lipid peroxides and increased the population of insulin-positive cells. Such results revealed differences between diabetogenic stimuli in determining the destiny of β-cells, emerging HG, H2O2, and STZ, but not cytokines, as contributing factors to ferroptosis and shed new light on an antidiabetic strategy based on Nrf2 activation. Thus, targeting ferroptosis in diabetes might be a promising new approach for preservation of the β-cell population. Our results obtained from in vivo study strongly justify this approach.

Cell death plays an important role in diabetes-induced liver dysfunction. Ferroptosis is a newly defined regulated cell death caused by iron-dependent lipid peroxidation. Our previous studies have shown that high glucose and streptozotocin (STZ) cause β-cell death through ferroptosis and that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, improves β-cell viability, islet morphology, and function. This study was aimed to examine in vivo the involvement of ferroptosis in diabetes-related pathological changes in the liver. For this purpose, male C57BL/6 mice, in which diabetes was induced with STZ (40 mg/kg/5 consecutive days), were treated with Fer-1 (1 mg/kg, from day 1–21 day). It was found that in diabetic mice Fer-1 improved serum levels of ALT and triglycerides and decreased liver fibrosis, hepatocytes size, and binucleation. This improvement was due to the Fer-1-induced attenuation of ferroptotic events in the liver of diabetic mice, such as accumulation of pro-oxidative parameters (iron, lipofuscin, 4-HNE), decrease in expression level/activity of antioxidative defense-related molecules (GPX4, Nrf2, xCT, GSH, GCL, HO-1, SOD), and HMGB1 translocation from nucleus into cytosol. We concluded that ferroptosis contributes to diabetes-related pathological changes in the liver and that the targeting of ferroptosis represents a promising approach in the management of diabetes-induced liver injury.

Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid‐laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (ie 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige‐like morphology and exhibited an intermediate gene expression profile, with both beige‐like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post‐transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (eg carbonyl cyanide‐4‐phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC‐derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown‐like features after 3 weeks of acclimatization at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible.

The main pathological hallmark of diabetes is the loss of functional

Temperature is a key regulator of brown adipose tissue (BAT) function, acting through central sensory inputs to influence metabolism and energy storage. Although animal models have produced a wealth of information on the pathways, effectors and responses mediating the physiological response of adipose tissue to temperature in vivo, the use of cell culture models now offers evidence of an additional cell-autonomous response to temperature changes, in the absence of neural input. In particular, stem cell models provide new insight into the regulation of adipogenic differentiation and the induction of browning features in vitro. Here the basis for adipogenic responsiveness to low temperature is discussed, together with different human cell models available to outline the benefits of cell-based approaches for future BAT research.

Brown adipose tissue (BAT) is able to rapidly generate heat and metabolise macronutrients, such as glucose and lipids, through activation of mitochondrial uncoupling protein 1 (UCP1). Diet can modulate UCP1 function but the capacity of individual nutrients to promote the abundance and activity of UCP1 is not well established. Caffeine consumption has been associated with loss of body weight and increased energy expenditure, but whether it can activate UCP1 is unknown. This study examined the effect of caffeine on BAT thermogenesis in vitro and in vivo. Stem cell-derived adipocytes exposed to caffeine (1 mM) showed increased UCP1 protein abundance and cell metabolism with enhanced oxygen consumption and proton leak. These functional responses were associated with browning-like structural changes in mitochondrial and lipid droplet content. Caffeine also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial biogenesis, together with a number of BAT selective and beige gene markers. In vivo, drinking coffee (but not water) stimulated the temperature of the supraclavicular region, which co-locates to the main region of BAT in adult humans, and is indicative of thermogenesis. Taken together, these results demonstrate that caffeine can promote BAT function at thermoneutrality and may have the potential to be used therapeutically in adult humans.

Background/aims Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. Methods Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine - MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). Results Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. Conclusion These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.

Brown and beige adipocytes are characterised as expressing the unique mitochondrial uncoupling protein (UCP)1 for which the primary stimulus in vivo is cold exposure. The extent to which cold-induced UCP1 activation can also be achieved in vitro, and therefore perform a comparable cellular function, is unknown. We report an in vitro model to induce adipocyte browning using bone marrow (BM) derived mesenchymal stem cells (MSC), which relies on differentiation at 32 °C instead of 37 °C. The low temperature promoted browning in adipogenic cultures, with increased adipocyte differentiation and upregulation of adipogenic and thermogenic factors, especially UCP1. Cells exhibited enhanced uncoupled respiration and metabolic adaptation. Cold-exposed differentiated cells showed a marked translocation of leptin to adipocyte nuclei, suggesting a previously unknown role for leptin in the browning process. These results indicate that BM-MSC can be driven to forming beige-like adipocytes in vitro by exposure to a reduced temperature. This in vitro model will provide a powerful tool to elucidate the precise role of leptin and related hormones in hitherto functions in the browning process.

The aim of this study was to determine the effects of long-term sucrose overfeeding on functional capacity and ultrastructural characteristics of the rat brown adipose tissue (BAT). For the study, 16 male Wistar rats, chow-fed and kept under standard laboratory conditions, were divided into 2 equal groups. The rats from a control group drank tap water, whereas those from a sucrose overfed group were allowed to drink 10% sucrose solution for 21 days. Structural changes of BAT were analysed at the level of light and electron microscopy on routinely prepared tissue sections or using immunohistochemical staining, in combination with stereological methods. Obtained results have shown that the significantly increased energy intake in sucrose overfed rats did not result in a higher gain of body mass compared with controls. The light microscopy analysis revealed that the BAT acquired the appearance of a thermogenically active tissue, with intensified vascularisation, reduced size of brown adipocytes and increased multilocularity. At the ultrastructural level, mitochondria of brown adipocytes became more abundant, enlarged and contained more cristae in comparison to control animals. The immunoexpression of uncoupling protein 1 (UCP1) and noradrenaline, as markers of BAT thermogenic status, was increased, whereas the pattern of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) was slightly modified. Taken together, the results of this investigation indicated that BAT possesses the ability to increase thermogenic capacity/activity in response to high energy intake and to prevent body mass gain. These findings are particularly relevant in view of recent reports on the existence of functional BAT in adult humans and its potential use to combat obesity.

Objective. Metabolic homeostasis depends on adipocyte metabolic responses/processes, most of which are redox-regulated. Besides, visceral and subcutaneous adipose tissues (VAT and SAT, respectively) differ metabolically and in their contribution to metabolic complications, but their redox characteristics in humans are still unknown. To understand the molecular mechanisms of metabolic syndrome development, we analysed the redox characteristics of VAT and SAT in groups with various body weights and metabolic risks. Material and Methods. Fifty premenopausal women were classified according to body mass index into normal-weight and obese groups, and these groups were further sub-classified into metabolically healthy and metabolically obese ({''}at risk{''}) based on the homeostasis model assessment of insulin resistance (HOMA-IR) index and the triglyceride, total-, LDL- and HDL-cholesterol levels. Antioxidant components, NADPH oxidase protein and 4-hydroxynonenal (4-HNE) levels were analysed in VAT and SAT. Results. Compared with the SAT, the VAT showed a higher basal level of glutathione (GSH) and GSH-dependent enzyme activities. Compared with the metabolically healthy normal-weight controls, the obese groups of women showed lower GSH levels in both depots. However, in these groups, additional prooxidative changes (increased NADPH oxidase and 4-HNE and decreased levels of SOD and/or CAT) were observed only in VAT. Conclusions. Because of the critical role of thiol-redox homeostasis in lipogenesis, interdepot-differences in the GSH-dependent antioxidant part may be connected to the higher metabolic activity found in VAT. Analogously, the lower GSH levels that occur during obesity and the corresponding additional redox imbalance may be signs of VAT metabolic dysfunction that underlie the subsequent metabolic impairment. (C) 2014 Elsevier Inc. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173054, 173055]

Context: Brown adipose tissue (BAT) has the unique ability of generating heat due to the expression of mitochondrial uncoupling protein 1 (UCP1). A recent discovery regarding functional BAT in adult humans has increased interest in the molecular pathways of BAT development and functionality. An important role for estrogen in white adipose tissue was shown, but the possible role of estrogen in human fetal BAT (fBAT) is unclear. Objective: The objective of this study was to determine whether human fBAT expresses estrogen receptor alpha (ER alpha) and ER beta. In addition, we examined their localization as well as their correlation with crucial proteins involved in BAT differentiation, proliferation, mitochondriogenesis and thermogenesis including peroxisome proliferator-activated receptor gamma (PPAR gamma), proliferating cell nuclear antigen (PCNA), PPAR gamma-coactivator-1 alpha (PGC-1 alpha), and UCP1. Design: The fBAT was obtained from 4 human male fetuses aged 15, 17, 20, and 23 weeks gestation. ER alpha and ER beta expression was assessed using Western blotting, immunohistochemistry, and immunocytochemistry. Possible correlations with PPAR gamma, PCNA, PGC-1 alpha, and UCP1 were examined by double immunofluorescence. Results: Both ER alpha and ER beta were expressed in human fBAT, with ER alpha being dominant. Unlike ER beta, which was present only in mature brown adipocytes, we detected ER alpha in mature adipocytes, preadipocytes, mesenchymal and endothelial cells. In addition, double immunofluorescence supported the notion that differentiation in fBAT probably involves ER alpha. Immunocytochemical analysis revealed mitochondrial localization of both receptors. Conclusion: The expression of both ER alpha and ER beta in human fBAT suggests a role for estrogen in its development, primarily via ER alpha. In addition, our results indicate that fBAT mitochondria could be targeted by estrogens and pointed out the possible role of both ERs in mitochondriogenesis. Serbian Ministry of Education, Science, and Technological Development {[}173055, 173054]

Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term l-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold. Animals (six per group) acclimated to room temperature (22 +/- A 1 A degrees C) and cold (4 +/- A 1 A degrees C), respectively, were treated with 2.25 \% l-arginine, a substrate for NOSs, or with 0.01 \% N (omega)-nitro-l-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression. Long-term dietary l-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine. Our results demonstrate that long-term dietary l-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP. Serbian Ministry of Education, Science and Technological Development {[}173055]

Hippocampal structural changes associated with diabetes-related cognitive impairments are well described, but their molecular background remained vague. We examined whether/how diabetes alters molecular basis of energy metabolism in hippocampus readily after diabetes onset, with special emphasis on its redox-sensitivity. To induce diabetes, adult Mill Hill hybrid hooded rats received a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were further divided in two subgroups receiving (i) or not (ii) superoxide dismutase (SOD) mimic, [Mn(II)(pyane)Cl2] for 7 days, i.p. Treatment of the diabetic animals started after blood glucose level ≥12 mM. Diabetes decreased protein levels of oxidative phosphorylation components: complex III and ATP synthase. In contrast, protein amounts of glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, and hypoxia-inducible factor-1α - the key regulator of energy metabolism in stress conditions, were higher in diabetic animals. Treatment with SOD mimic restored/increased the levels of oxidative phosphorylation components and returned hypoxia-inducible factor-1α to control level, while diabetes-induced up-regulation of glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, was additionally stimulated. To conclude, our results provide insight into the earliest molecular changes of energy-producing pathways in diabetes that may account for structural/functional disturbance of hippocampus, seen during disease progression. Also, data suggest [Mn(II)(pyane)Cl2] as potential therapeutic agent in cutting-edge approaches to threat this widespread metabolic disorder.

The stereological structure of rat adrenal gland was analysed by light and electron microscopy after an acute (60 min) exposure to high ambient temperature (38°C). Under these conditions a significant increase in plasma corticotrophin (ACTH), serum corticosterone and aldosterone levels were observed. Histological and stereological investigation at light microscopy showed significant decrease in volume density of capsule and zona glomerulosa, increase in volume of fasciculata cells, and decrease of numerical density of zona fasciculata cells and mean diameter of blood vessels. At the ultrastructural level, volume density of nuclei and mitochondria of zona glomerulosa cells were significantly increased and that of lipid droplets decreased. Volume density of mitochondria of fasciculata cells was significantly increased, while number of lipid droplets per µm2 of cell was reduced. In the cells of zona reticularis significant increase in the number of lipid droplets was found. The response of zona glomerulosa may be interpreted as immediate reaction to dehydration, while alterations detected in zona fasciculata, which were less extensive, were related to purely stressogenic effects of high ambiental temperature.

Background/Aims: Study elucidates and compares the mitochondrial bioenergetic-related molecular basis of sevoflurane and propofol cardioprotection during aortic valve replacement surgery due to aortic valve stenosis. Methods: Twenty-two patients were prospectively randomized in two groups regarding the anesthetic regime: sevoflurane and propofol. Hemodynamic parameters, biomarkers of cardiac injury and brain natriuretic peptide (BNP) were measured preoperatively and postoperatively. In tissue samples, taken from the interventricular septum, key mitochondrial molecules were determined by Western blot, real time PCR, as well as confocal microscopy and immunohisto- and immunocyto-chemical analysis. Results: The protein levels of cytochrome c oxidase and ATP synthase were higher in sevoflurane than in propofol group. Nevertheless, cytochrome c protein content was higher in propofol than sevoflurane receiving patients. Propofol group also showed higher protein level of connexin 43 (Cx43) than sevoflurane group. Besides, immunogold analysis showed its mitochondrial localization. The mRNA level of mtDNA and uncoupling protein (UCP2) were higher in propofol than sevoflurane patients, as well. On the other hand, there were no significant differences between groups in hemodynamic assessment, intensive care unit length of stay, troponin I and BNP level. Conclusions: Our data indicate that sevoflurane and propofol lead to cardiac protection via different mitochondrially related molecular mechanisms. It appears that sevoflurane acts regulating cytochrome c oxidase and ATP synthase, while the effects of propofol occur through regulation of cytochrome c, Cx43, mtDNA transcription and UCP2. Copyright (C) 2012 S. Karger AG, Basel Ministry of Education and Science [173055, 173054]

The function of pancreatic ?-cells is to produce and secrete insulin, a crucial hormone in carbohydrate metabolism. The transcription factor PDX1 is required for insulin gene transcription and mature ?-cell function. Since this factor is regulated by triiodothyronine, a disturbance in insulin biosynthesis and/or secretion is usually related to a deficiency of this hormone. In the present study, we used methods of immunohistochemistry, stereology and electron microscopy to explore the correlation between altered thyroid status and insulin synthesis/secretion in a model of methimazole-induced hypothyroidism in rats. In hypothyroid animals fewer functional PDX1-positive ?-cells were detected in the islets of Langerhans, while insulin immunostaining was stronger. Stereological analysis of ?-cell granules revealed more numerous immature insulin granules in hypothyroid rats. Taken together, these data suggest that the applied treatment caused impaired insulin synthesis and secretion. Rare cells with granules characteristic for both ?- and ?-cells observed in hypothyroid animals could provide functional compensation for diminished insulin synthesis.

An ultramicroscopic study of brown adipose tissue (BAT) of rats treated with Ca-SANDOZ? (480 mg/l) for 3 days, revealed erythrocyte exovesiculation and migratory erythrocytic complexes from the capillaries to adipocyte cytoplasm and mitochondria. Two types of erythrocytic material transfer were observed: (i) numerous exocytic vesicles with electron dense material leaving the erythrocytes; (ii) furcated complexes with microholes, embedded in amorphous material. The content of red blood cell (RBC) complexes passed through the capillaries and transferred to the brown adipocytes where it was detectable in the cytoplasm and mitochondria. Light microscopy confirmed sphenoechinocytic transformation of the RBCs in the blood smears of the Ca-SANDOZ? treated rats.

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 (R) treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ (R) 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 (R) 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 (R) 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 (R) stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes. Serbian Ministry of Education, Science and Technological Development {[}173055, 173054]

The aim of this study was to investigate lipofuscin origin in brown adipocytes of hyperinsulinaemic rats and the possible role of lipid peroxidation and iron in this process. Ultrastructural examination revealed hyperinsulinaemia-induced enhancement in the lipofuscin production, accompanied by an increase of mitochondrial damage in brown adipocytes. Extensive fusions of lipid droplets and mitochondria with lysosomes were also observed. Confocal microscopy showed lipofuscin autofluorescence emission in brown adipose tissue (BAT) after excitation at 488 nm and 633 nm, particularly in the insulin-treated groups. The presence and distribution of lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE), in brown adipocytes was assessed by immunohistochemical examination revealing its higher content after treatment with insulin. The iron content was quantified by electron dispersive X-ray analysis (EDX) showing its higher content in the hyperinsulinaemic groups. The ultrastucture of the majority of lipofuscin granules suggests their mitochondrial origin, which was additionally confirmed by their co-localization with ATP synthase. In conclusion, our results suggest that increased lipofuscinogenesis in the brown adipocytes of hyperinsulinaemic rats is a consequence of lipid peroxidation, mitochondrial damage and iron accumulation.

Mitochondria play an important role in sperm cell maturation and function. Here, we examined whether (and how) changes in sperm redox milieu affect the functional status of sperm mitochondria, that is, sperm functionality. Compared with the control, incubation in Tyrode's medium for 3 h, under noncapacitating conditions, decreased sperm motility, the amount of nitric oxide ((NO)-N-center dot), the number of MitoTracker (R) Green FM (MT-G) positive mitochondria, and the expression of complexes I and IV of the mitochondrial respiratory chain. In turn, superoxide dismutase (SOD) mimic (M40403) treatment restored/increased these parameters, as well as the expression of endothelial nitric oxide synthase, manganese SOD, and catalase. These data lead to the hypothesis that M40403 improves mitochondrial functional state and motility of spermatozoa, as well as (NO)-N-center dot might be involved in the observed effects of the mimic. Antioxid. Redox Signal. 18, 170-178. Ministry of Education and Science of the Republic of Serbia [173054]; University of Erlangen-Nuremberg

Any alteration in oxidative metabolism is coupled with a corresponding response by an antioxidant defense (AD) in appropriate subcellular compartments. Seasonal hibernators pass through circannual metabolic adaptations that allow them to either maintain euthermy (cold acclimation) or enter winter torpor with body temperature falling to low values. The present study aimed to investigate the corresponding pattern of AD enzyme protein expressions associated with these strategies in the main tissues involved in whole animal energy homeostasis: brown and white adipose tissues (BAT and WAT, respectively), liver, and skeletal muscle. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 +/- 1 C) and then divided into two groups: (1) animals fell into torpor (hibernating group) and (2) animals stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). We examined the effects of cold acclimation and hibernation on the tissue-dependent protein expression of four enzymes which catalyze the two-step detoxification of superoxide to water: superoxide dismutase 1 and 2 (SOD 1 and 2), catalase (CAT), and glutathione peroxidase (GSH-Px). The results showed that hibernation induced an increase of AD enzyme protein expressions in BAT and skeletal muscle. However, AD enzyme contents in liver were largely unaffected during torpor. Under these conditions, different WAT depots responded by elevating the amounts of specific enzymes, as follows: SOD 1 in retroperitoneal WAT, GSH-Px in gonadal WAT, and CAT in subcutaneous WAT. Similar perturbations of AD enzymes contents were seen in all tissues during cold acclimation, often in a time-dependent manner. It can be concluded that BAT and muscle AD capacity undergo the most dramatic changes during both cold acclimation and hibernation, while liver is relatively unaffected by either condition. Additionally, this study provides a basis for further metabolic study that will illuminate the causes of these tissue-specific AD responses, particularly the novel finding of distinct responses by different WAT depots in hibernators. (C) 2013 Elsevier Inc. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia [173055]

This study examined the molecular basis of energy-related regulatory mechanisms underlying metabolic recruitment of skeletal muscle during cold acclimation and possible involvement of the L-arginine/nitric oxide-producing pathway. Rats exposed to cold (4 +/- 1 degrees C) for periods of 1, 3, 7, 12, 21 and 45 days were divided into three groups: untreated, L-arginine treated and N-omega-nitro-L-arginine methyl ester (L-NAME) treated. Compared with controls (22 +/- 1 degrees C), there was an initial increase in the protein level of 5'-AMP-activated protein kinase alpha (day 1), followed by an increase in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) and peroxisome proliferator-activated receptors (PPARs): PPAR alpha and PPAR gamma from day 1 and PPAR delta from day 7 of cold acclimation. Activation of the PGC-1 alpha/PPAR transcription program was accompanied by increased protein expression of the key metabolic enzymes in beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation, with the exceptions in complex I (no changes) and ATP synthase (decreased at day 1). Cold did not affect hexokinase and GAPDH protein levels, but increased lactate dehydrogenase activity compared with controls (1-45 days). L-arginine sustained, accelerated and/or intensified cold-induced molecular remodeling throughout cold acclimation. L-NAME exerted phase-dependent effects: similar to L-arginine in early cold acclimation and opposite after prolonged cold exposure (from day 21). It seems that upregulation of the PGC-1 alpha/PPAR transcription program early during cold acclimation triggers the molecular recruitment of skeletal muscle underlying the shift to more oxidative metabolism during prolonged cold acclimation. Our results suggest that nitric oxide has a role in maintaining the skeletal muscle oxidative phenotype in late cold acclimation but question its role early in cold acclimation. Ministry of Education, Science and Technological development of the Republic of Serbia [173054, 173055]

The expression profiles of adiponectin, resistin, 5′-AMP-activated protein kinaseα(AMPKα), hypoxia-inducible factor-1α(HIF-1α), and key enzymes of glucose and fatty acid metabolism and oxidative phosphorylation in rat retroperitoneal white adipose tissue (RpWAT) during 45-day cold acclimation were examined. After transient suppression on day 1, adiponectin protein level increased following sustained cold exposure. In parallel, on day 1, the protein level of HIF-1αwas strongly induced and AMPKαsuppressed, while afterwards the reverse was seen. What is more, after an initial decrease on day 1, a sequential increase in pyruvate dehydrogenase, acyl-CoA dehydrogenase, cytochromecoxidase, and ATP synthase and a decrease in acetyl-CoA carboxylase (from day 3) were observed. Similar to adiponectin, protein level of resistin showed a biphasic profile: it increased after days 1, 3, and 7 and decreased below the control after 21 days of cold-acclimation. In summary, the data suggest that adiponectin and resistin are important integrators of RpWAT metabolic response and roles it plays during cold acclimation. It seems that AMPKαmediate adiponectin effects on metabolic remodeling RpWAT during cold acclimation.

In this study, the effects of L-arginine-nitric-oxide ((∙)NO)-producing pathway on protein content of ubiquitin, as an important component of ubiquitin-proteasome system for protein removal, were investigated. We showed that L-arginine markedly decreased ubiquitin protein content in interscapular brown adipose tissue, both in thermogenic inactive (at room temperature) and thermogenic active (on cold) states; while in L-NAME-treated groups this effect was abolished. This result suggests that nitric oxide ((∙)NO), besides well established roles, is involved in this aspect of structure remodeling, as well.

Background Brown adipose tissue thermogenic program includes complex molecular and structural changes. However, energetic aspects of this process are poorly depicted. Methods We investigated time-dependent reprogramming of interscapular brown adipose tissue (IBAT) energy metabolism during cold-acclimation, as well as the effects of nitric oxide ( NO) on those changes. Rats were exposed to cold (4 ± 1 °C) for periods of 1, 3, 7, 12, 21, and 45 days, and divided into three groups: control, treated with L-arginine, and treated with Nω-nitro-L-arginine methyl ester (L-NAME). Results In the early phase of cold-acclimation (up to 7 days), the protein levels of all metabolic parameters and oxidative phosphorylation components were below the control. However, metabolic parameters and respiratory chain components entered a new homeostatic level in the late phase of cold-acclimation. These changes were accompanied with increased protein levels of phospho-AMP-dependent protein kinase-α (phospho-AMPKα) on the first day of cold-acclimation, and hypoxia-inducible factor-1α (HIF-1α) throughout early cold-acclimation. L-arginine positively affected protein expression of enzymes involved in glucose metabolism and β-oxidation of fatty acids in the early phase of cold-acclimation, and oxidative phosphorylation components throughout cold-acclimation. In contrast, L-NAME had the opposite effects. Conclusion Results suggest that IBAT structural remodeling is followed by energy metabolism reprogramming, which control might be orchestrated by the action of AMPKα and HIF-1α. Data also indicated the involvement of L-arginine- NO in the regulation of IBAT metabolism. General significance Results obtained in this study might be of great importance for elucidating regulatory pathways governing energy metabolism in both physiological and pathophysiological states.

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 chemiluminescence's 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.

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.

Developmental dysfunction in embryos, such as a lethal level of fragmentation, is assumed to be mitochondrial in origin. This study investigated the molecular basis of mitochondrial impairment in embryo fragmentation. Transcription patterns of factors that determine mitochondrial functionality: (i) components of the oxidative phosphorylation (OXPHOS) – complex I, cytochrome b, complex IV and ATP synthase; (ii) mitochondrial membrane potential (MMP); (iii) mitochondrial DNA (mtDNA) content and (iv) proteins involved in mitochondrial dynamics, mitofusin 1 (Mfn1) and dynamin related protein 1 (Drp1) were examined in six-cells Day 3 non-fragmented (control), low-fragmented (LF) and high-fragmented (HF) human embryos. Gene expression of mitochondria-encoded components of complex I and IV, cytochrome b and mtDNA were increased in HF embryos compared with control and LF embryos. In LF embryos, expression of these molecules was decreased compared with control and HF embryos. Both classes of fragmented embryos had decreased MMP compared with control. LF embryos had increased gene expression of Mfn1 accompanied by decreased expression of Drp1, while HF embryos had decreased Mfn1 expression but increased Drp1 expression. The study revealed that each improper transcriptional (in)activation of mitochondria-encoded components of the OXPHOS during early in vitro embryo development is associated with a decrease in MMP and with embryo fragmentation. The results also showed the importance of mitochondrial dynamics in fragmentation, at least in the extent of this process.

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.