Nataša Veličković

Tissue-specific regulation of inflammation by macrophage migration inhibitory factor and glucocorticoids in fructose-fed Wistar rats.

High fructose consumption is commonly associated with insulin resistance, disturbed glucose homeostasis and low-grade inflammation. Increased glucocorticoid production within adipose tissue has been implicated in the pathogenesis of fructose-induced metabolic syndrome. Immunosuppressive actions of glucocorticoids can be counter-regulated by macrophage migration inhibitory factor (MIF), which is recognised as a key molecule in metabolic inflammation. In the present study, we hypothesised that coordinated action of glucocorticoids and MIF can mediate the effects of a high-fructose diet on adipose tissue and liver inflammation. We examined the effects of long-term consumption of a 10% fructose solution on corticosterone (CORT) and MIF levels in rat blood plasma, liver and adipose tissue, as well as MIF and TNF-a mRNA expression and NF-kB activation in the same tissues. The high-fructose diet led to an increase in both CORT and MIF in the adipose tissue, and a highly significant positive correlation between their levels was observed. The attenuated NF-kB activation and unaltered TNF-a mRNA expression noticed in the adipose tissue could be interpreted as an outcome of the opposing actions of CORT and MIF. In contrast to adipose tissue, inflammation in the liver was characterised by NF-kB activation, an increased TNF-a mRNA level and unchanged levels of MIF protein, MIF mRNA and CORT. Overall, these findings suggest that a high-fructose diet differently affects the levels of glucocorticoids and MIF in the adipose tissue and liver, implicating that fructose over-consumption has tissue-specific effects on regulation of metabolic inflammation.

Fructose consumption enhances glucocorticoid action in rat visceral adipose tissue.

The rise in consumption of refined sugars high in fructose appears to be an important factor for the development of obesity and metabolic syndrome. Fructose has been shown to be involved in genesis and progression of the syndrome through deregulation of metabolic pathways in adipose tissue. There is evidence that enhanced glucocorticoid regeneration within adipose tissue, mediated by the enzyme 11beta-hydroxysteroid dehydrogenase Type 1 (11βHSD1), may contribute to adiposity and metabolic disease. 11βHSD1 reductase activity is dependent on NADPH, a cofactor generated by hexose-6-phosphate dehydrogenase (H6PDH). We hypothesized that harmful effects of long-term high fructose consumption could be mediated by alterations in prereceptor glucocorticoid metabolism and glucocorticoid signaling in the adipose tissue of male Wistar rats. We analyzed the effects of 9-week drinking of 10% fructose solution on dyslipidemia, adipose tissue histology and both plasma and tissue corticosterone level. Prereceptor metabolism of glucocorticoids was characterized by determining 11βHSD1 and H6PDH mRNA and protein levels. Glucocorticoid signaling was examined at the level of glucocorticoid receptor (GR) expression and compartmental redistribution, as well as at the level of expression of its target genes (GR, phosphoenolpyruvate carboxyl kinase and hormone-sensitive lipase). Fructose diet led to increased 11βHSD1 and H6PDH expression and elevated corticosterone level within the adipose tissue, which was paralleled with enhanced GR nuclear accumulation. Although the animals did not develop obesity, nonesterified fatty acid and plasma triglyceride levels were elevated, indicating that fructose, through enhanced prereceptor metabolism of glucocorticoids, could set the environment for possible later onset of obesity.

High-fructose diet leads to visceral adiposity and hypothalamic leptin resistance in male rats — do glucocorticoids play a role?

Fructose overconsumption has been involved in the genesis and progression of the metabolic syndrome. Hypothalamus and adipose tissue, major organs for control of food intake and energy metabolism, play crucial roles in metabolic homeostasis. We hypothesized that glucocorticoid signaling mediates the effects of a fructose-enriched diet on visceral adiposity by acting on neuropeptide Y (NPY) in the hypothalamus and altering adipogenic transcription factors in the visceral adipose tissue. We analyzed the effects of 9-week consumption of 60% fructose solution on dyslipidemia, insulin and leptin sensitivity, and adipose tissue histology in male Wistar rats. Glucocorticoid signaling was assessed in both hypothalamus and visceral adipose tissue, while the levels of peroxisome-proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding protein-1 (SREBP-1) and lipin-1, together with the levels of their target genes expression, were analyzed in the visceral adipose tissue. The results showed that long-term consumption of highly concentrated liquid fructose led to the development of visceral adiposity, elevated triglycerides and hypothalamic leptin resistance accompanied by stimulated glucocorticoid signaling and NPY mRNA elevation. Results from adipose tissue implied that fructose consumption shifted the balance between glucocorticoid receptor and adipogenic transcriptional factors (PPARγ, SREBP-1 and lipin-1) in favor of adipogenesis judged by distinctly separated populations of small adipocytes observed in this tissue. In summary, we propose that high-fructose-diet-induced alterations of glucocorticoid signaling in both hypothalamus and adipose tissue result in enhanced adipogenesis, possibly serving as an adaptation to energy excess in order to limit deposition of fat in nonadipose tissues.

The impact of different fructose loads on insulin sensitivity, inflammation, and PSA-NCAM-mediated plasticity in the hippocampus of fructose-fed male rats

Abstract Objectives High fructose diet has been shown to have damaging effects on the hippocampus, a brain region critical for learning and memory. Fructose-induced hippocampal dysfunction may arise from insulin resistance and inflammation, and from concomitant changes in plasticity-related presynaptic proteins. We hypothesized that long-term access to fructose (10% and 60% solutions over a period of 9 weeks) affects insulin sensitivity, hippocampal inflammation, and synaptic plasticity in male Wistar rats. Methods We used the area under curve (AUC) glucose value and inhibitory Ser307 phosphorylation of hippocampal insulin receptor substrate 1 (IRS-1) as hallmarks of insulin resistance. To examine inflammatory state, we analysed protein levels and intracellular redistribution of glucocorticoid receptor and nuclear factor-κB (NFκB), as well as mRNA levels of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Polysialylated neural cell adhesion molecule (PSA-NCAM) protein was used as a synaptic plasticity marker. Results The results indicate different impacts of diverse fructose-enriched diets on insulin sensitivity and hippocampal inflammation and plasticity. Long-term ingestion of 10% fructose solution led to increase in AUC glucose value, as well as to elevation in hippocampal IRS-1 Ser307 phosphorylation and increase in IL-6 mRNA. In rats consuming 60% fructose, the level of PSA-NCAM was reduced, in parallel with augmented glucocorticoid signalization. Discussion The results showed that long-term consumption of 10% fructose solution induces hippocampal insulin resistance and inflammation, with no concomitant plasticity changes. Interestingly, rats fed with higher concentrations of fructose displayed impaired plastic response of the hippocampus, coinciding with augmented glucocorticoid signalling, which may provide a basis for cognitive deficits associated with metabolic syndrome.

Low-Dose Dexamethasone Treatment Promotes the Pro-Survival Signalling Pathway in the Adult Rat Prefrontal Cortex

Synthetic glucocorticoid dexamethasone (DEX), a highly potent anti‐inflammatory and immunosuppressive agent, is widely used in the treatment of brain cancer, as well as for inflammatory and autoimmune diseases. The present study aimed to determine whether low‐dose subchronic DEX treatment (100 μg/kg for eight consecutive days) exerts long‐term effects on apoptosis in the adult rat prefrontal cortex (PFC) by examining the expression of cell death‐promoting molecules [poly(ADP‐ribose) polymerase (PARP), p53, procaspase 3, cleaved caspase 3, Bax] and cell‐survival molecules (AKT, Bcl‐2). The results obtained revealed that body, thymus and adrenal gland weights, as well corticosterone levels, in the serum and PFC were reduced 1 day after the last DEX injection. In the PFC, DEX caused activation of AKT, augmentation of pro‐survival Bcl‐2 protein and an enhanced Bcl‐2/Bax protein ratio, as well Bcl‐2 translocation to the mitochondria. An unaltered profile with respect to the protein expression of apoptotic molecules PARP, procaspase 3 and Bax was detected, whereas p53 protein was decreased. Reverse transcriptase ‐polymerase chain reaction analysis showed a decrease of p53 mRNA levels and no significant difference in Bcl‐2 and Bax mRNA expression in DEX‐treated rats. Finally, a DNA fragmentation assay and Fluoro‐Jade staining demonstrated no considerable changes in apoptosis in the rat PFC. Our findings support the concept that low‐dose DEX creates a hypocorticoid state in the brain and also indicate that subchronic DEX treatment activates the pro‐survival signalling pathway but does not change apoptotic markers in the rat PFC. This mechanism might be relevant for the DEX‐induced apoptosis resistance observed during and after chemotherapy of patients with brain tumours.

Radiation-Induced Hyposuppression of the Hypothalamic-Pituitary-Adrenal Axis is Associated with Alterations of Hippocampal Corticosteroid Receptor Expression

Abstract Veličković, N., Djordjević, A., Matić, G. and Horvat, A. Radiation-Induced Hyposuppression of the Hypothalamic-Pituitary-Adrenal Axis is Associated with Alterations of Hippocampal Corticosteroid Receptor Expression. Radiat. Res. 169, 397–407 (2008). Therapeutic brain irradiation in children can cause a progressive decline in cognitive functions through a diminished capability to learn and memorize. Because of the known involvement of the hippocampus in memory consolidation, this study was aimed at examining the late effects of γ radiation on hypothalamic-pituitary-adrenal (HPA) axis activity and hippocampal corticosteroid receptor expression in an animal model of cranial radiotherapy. In the late-response phase, the basal and stress-induced corticosterone levels were not affected by radiation, but the suppression of glucocorticoid negative feedback by dexamethasone was attenuated in irradiated rats. Western blot analyses showed that exposure to radiation led to a decrease of cytosolic glucocorticoid receptor (GR) levels and a concomitant elevation of mineralocorticoid receptor (MR). The results obtained were complemented by those of RT-PCR, since the ratio of GR/MR mRNA was also decreased after radiation exposure. Dexamethasone appeared to be much less effective in shifting GR to the nuclear compartment in irradiated rats than in sham-irradiated animals. However, the expression of chaperones that aid GR intracellular trafficking, Hsp90 and Hsp70, remained unaffected. In conclusion, our data suggest that the hallmark of the late response to γ radiation is a hyposuppressive state of the HPA axis that is associated with a decrease in both the GR/MR ratio and the nuclear accumulation of dexamethasone-activated GR in the hippocampus.

Enhanced prereceptor glucocorticoid metabolism and lipogenesis impair insulin signaling in the liver of fructose-fed rats

Overconsumption of fructose, as a highly lipogenic sugar, may profoundly affect hepatic metabolism and has been associated with many components of the metabolic syndrome, particularly with insulin resistance and Type 2 diabetes. In this study, we proposed that high fructose diet may enhance lipogenesis and decrease insulin sensitivity in the liver through dysregulation of glucocorticoid signaling. Therefore, we examined the effects of long-term consumption of 10% fructose solution on triglyceridemia, liver histology and intracellular corticosterone level, as well as on 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and hexose-6-phosphate dehydrogenase (H6PDH) mRNA and protein levels in the rat liver. Glucocorticoid action was assessed by glucocorticoid receptor (GR) expression and intracellular redistribution. We also analyzed the expression of enzymes involved in gluconeogenesis and lipogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and lipin-1. The results have shown that fructose-rich diet led to increase in 11βHSD1 and H6PDH protein levels, while hepatic corticosterone concentration remained unchanged. Concomitantly, GR was increasingly accumulated in the cytoplasm, whereas its nuclear level was unchanged and accompanied by diminished PEPCK mRNA level. Elevation of lipin-1 in the liver microsomes suggested that fructose diet led to an increase in lipogenesis and consequently to hypertriglyceridemia. The observed increase of insulin receptor supstrate-1 phosphorylation on Ser(307) represents a hallmark of impaired insulin signaling in the liver of fructose-fed rat and probably is a consequence of the alterations in 11βHSD1 and lipin-1 levels. Overall, our findings suggest that fructose-rich diet may perturb hepatic prereceptor glucocorticoid metabolism and lipogenesis, resulting in hypertriglyceridemia and attenuated hepatic insulin sensitivity.

Possible involvement of glucocorticoids in 5α-dihydrotestosterone-induced PCOS-like metabolic disturbances in the rat visceral adipose tissue

Polycystic ovary syndrome (PCOS) is a reproductive and metabolic disorder characterized by hyperandrogenism, ovulatory dysfunction, visceral obesity and insulin resistance. We hypothesized that changes in glucocorticoid metabolism and signaling in the visceral adipose tissue may contribute to disturbances of lipid metabolism in the rat model of PCOS obtained by 5α-dihydrotestosterone (DHT) treatment of prepubertal female Wistar rats. The results confirmed that DHT treatment caused anovulation, obesity and dyslipidemia. Enhanced glucocorticoid prereceptor metabolism, assessed by elevated intracellular corticosterone and increased 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein levels, was accompanied by glucocorticoid receptor (GR) nuclear accumulation. In concert with the increased expression of GR-regulated prolipogenic genes (lipin-1, sterol regulatory element binding protein 1, fatty acid synthase, phosphoenolpyruvate carboxykinase), histological analyses revealed hypertrophic adipocytes. The results suggest that glucocorticoids influence lipid metabolism in the visceral adipose tissue in the way that may contribute to pathogenesis of metabolic disturbances associated with PCOS.

Effect of acute stress on NTPDase and 5'-nucleotidase activities in brain synaptosomes in different stages of development.

The aim of the present study was to examine the effect of acute restraint stress on rat brain synaptosomal plasma membrane (SPM) ecto‐nucleotidase activities at specific stages of postnatal development (15‐, 30‐, 60‐ and 90‐day‐old rats) by measuring the rates of ATP, ADP and AMP hydrolysis 1, 24 and 72 h post‐stress. At 1 h after stress NTPDase and ecto‐5′‐nucleotidase activities were decreased in rats aged up to 60 days old. In adult rats elevated enzyme activities were detected, which indicated the existence of different short‐term stress responses during development. A similar pattern of ATP and ADP hydrolysis changes as well as the ATP/ADP ratio in all developmental stages indicated that NTPDase3 was acutely affected after stress. The long‐term effect of acute stress on NTPDase activity differed during postnatal development. In juvenile animals (15 days old) NTPDase activity was not altered. However, in later developmental stages (30 and 60 days old rats) NTPDase activity decreased and persisted for 72 h post‐stress. In adult rats only ATP hydrolysis was decreased after 24 h, indicating that ecto‐ATPase was affected by stress. Ecto‐5′‐nucleotidase hydrolysing activity was decreased within 24 h in adult rats, while in 15‐ and 30‐day old rats it decreased 72 h post‐stress. At equivalent times in pubertal rats (60 days old) a slight activation of ecto‐5′‐nucleotidase was detected. Our results highlight the developmental‐dependence of brain ecto‐nucleotidase susceptibility to acute stress and the likely existence of different mechanisms involved in time‐dependent ecto‐nucleotidase activity modulation following stress exposure. Clearly there are differences in the response of the purinergic system to acute restraint stress between young and adult rats.

Long-term fructose-enriched diet introduced immediately after weaning does not induce oxidative stress in the rat liver

Increased fructose consumption is correlated with the rising prevalence of obesity, metabolic syndrome, and type 2 diabetes. It is believed that reactive oxygen species contribute to the development and progression of metabolic disturbances, especially those associated with insulin resistance. Dietary fructose produces both pro-oxidative and antioxidative effects, depending upon the experimental conditions, dosage, duration of treatment, and pathophysiological milieu. The effects of fructose overconsumption on young populations, which have an increased risk of developing metabolic disorders in adulthood, have not been fully elucidated. We have previously shown that rats subjected to a long-term fructose-enriched diet immediately after weaning display impaired hepatic insulin sensitivity. In this study, we tested the hypothesis that long-term fructose consumption induces alterations in the redox setting of the liver. Starting from the 21st day after birth, male Wistar rats were maintained for 9 weeks on a standard diet (control) or a fructose-enriched diet that consisted of standard food and 10% fructose solution instead of drinking water. The expression and activity of antioxidant enzymes as well as lipid peroxidation and protein damage markers were measured. The results showed that a fructose-enriched diet led to an increased expression of mitochondrial manganese superoxide dismutase but did not affect antioxidant enzymes activity, lipid peroxidation, thiol content, and the level of protein oxidation. Therefore, our results suggest that the decrease in hepatic insulin sensitivity that was previously observed in rats that were kept on the same diet regime might be attributed to molecular mechanisms other than redox disbalance. A possible fructose-related micronutrient deficiency should be examined.