Maria Elena Giusepponi

Regulation of hypothalamic neuropeptides gene expression in diet induced obesity resistant rats: possible targets for obesity prediction?

Several factors play a role in obesity (i.e., behavior, environment, and genetics) and epigenetic regulation of gene expression has emerged as a potential contributor in the susceptibility and development of obesity. To investigate the individual sensitivity to weight gain/resistance, we here studied gene transcription regulation of several hypothalamic neuropeptides involved in the control of energy balance in rats developing obesity (diet-induced obesity, DIO) or not (diet resistant, DR), when fed with a high fat diet. Rats have been followed up to 21 weeks of high fat diet exposure. After 5 weeks high fat diet exposure, the obese phenotype was developed and we observed a selective down-regulation of the orexigenic neuropeptide Y (NPY) and peroxisome proliferator-activated receptor gamma (PPAR-γ) genes. No changes were observed in the expression of the agouti-related protein (AgRP), as well as for all the anorexigenic genes under study. After long-term high fat diet exposure (21 weeks), NPY and PPAR-γ, as well as most of the genes under study, resulted not be different between DIO and DR, whereas a lower expression of the anorexigenic pro-opio-melanocortin (POMC) gene was observed in DIO rats when compared to DR rats. Moreover we observed that changes in NPY and POMC mRNA were inversely correlated with gene promoters DNA methylation. Our findings suggest that selective alterations in hypothalamic peptide genes regulation could contribute to the development of overweight in rats and that environmental factor, as in this animal model, might be partially responsible of these changes via epigenetic mechanism.

Epigenetic regulation of nociceptin/orphanin FQ and corticotropin-releasing factor system genes in frustration stress-induced binge-like palatable food consumption

Evidence suggests that binge eating may be caused by a unique interaction between dieting and stress. We developed a binge‐eating model in which female rats with a history of intermittent food restriction show binge‐like palatable food consumption after a 15‐minute exposure to the sight of the palatable food (frustration stress). The aim of the present study was to investigate the regulation of the stress neurohormone corticotropin‐releasing factor (CRF) system and of the nociceptin/orphanin FQ (N/OFQ) system genes in selective rat brain regions, using our animal model. Food restriction by itself seems to be responsible in the hypothalamus for the downregulation on messenger RNA levels of CRF‐1 receptor, N/OFQ and its receptor (NOP). For the latter, this alteration might be due to selective histone modification changes. Instead, CRF gene appears to be upregulated in the hypothalamus as well as in the ventral tegmental area only when rats are food restricted and exposed to frustration stress, and, of relevance, these changes appear to be due to a reduction in DNA methylation at gene promoters. Moreover, also CRF‐1 receptor gene resulted to be differentially regulated in these two brain regions. Epigenetic changes may be viewed as adaptive mechanisms to environmental perturbations concurring to facilitate food consumption in adverse conditions, that is, in this study, under food restriction and stressful conditions. Our data on N/OFQ and CRF signaling provide insight on the use of this binge‐eating model for the study of epigenetic modifications in controlled genetic and environmental backgrounds.

Hypothalamic expression of inflammatory mediators in an animal model of binge eating

HighlightsFood‐restricted rats stressed during non‐estrus develop binge eating (BE) behavior.BE is associated to changes in mRNA levels of inflammatory markers in hypothalamus.INOS mRNA increases in the anterior‐tuberal hypothalamus of BE rats.IL‐18/IL‐18R system is down‐regulated in hypothalamus of BE rats. ABSTRACT Binge eating episodes are characterized by uncontrollable, distressing eating of a large amount of highly palatable food and represent a central feature of bingeing related eating disorders. Research suggests that inflammation plays a role in the onset and maintenance of eating‐related maladaptive behavior. Markers of inflammation can be selectively altered in discrete brain regions where they can directly or indirectly regulate food intake. In the present study, we measured expression levels of different components of cytokine systems (IL‐1, IL‐6, IL‐18, TNF‐&agr; and IFN‐&ggr;) and related molecules (iNOS and COX2) in the preoptic and anterior‐tuberal parts of the hypothalamus of a validated animal model of binge eating. In this animal model, based on the exposure to both food restriction and frustration stress, binge‐like eating behavior for highly palatable food is not shown when animals are exposed to the frustration stress during the estrus phase. We found a characteristic down‐regulation of the IL‐18/IL‐18 receptor system (with increased expression of the inhibitor of the pro‐inflammatory cytokine IL‐18, IL‐18BP, together with a decreased expression of the binding chain of the IL‐18 receptor) and a three‐fold increase in the expression of iNOS specifically in the anterior‐tuberal region of the hypothalamus of animals that develop a binge‐like eating behavior. Differently, when food restricted animals were stressed during the estrus phase, IL‐18 expression increased, while iNOS expression was not significantly affected. Considering the role of this region of the hypothalamus in controlling feeding related behavior, this can be relevant in eating disorders and obesity. Our data suggest that by targeting centrally selected inflammatory markers, we may prevent that disordered eating turns into a full blown eating disorder.

Hypothalamic CRF1 receptor mechanisms are not sufficient to account for binge‐like palatable food consumption in female rats

OBJECTIVE The present study evaluated the effect of systemic injection of the CRF1 receptor antagonist R121919, the corticosterone synthesis inhibitor metyrapone and central amygdala (CeA) injections of the nonselective CRF antagonist D-Phe-CRF(12-41) in rats in which binge eating was evoked by stress and cycles of food restriction. METHOD Female rats were subjected or not to repeated cycles of regular chow food restriction/ad libitum feeding during which they were also given limited access (2 h) to palatable food. On the test day, rats were either exposed or not to the sight of the palatable food for 15 min without allowing access, before assessing food consumption. RESULTS Systemic injections of R121919, but not of the metyrapone, blocked binge-like eating behavior. Restricted and stressed rats showed up-regulation of crh1 receptor mRNA signal in the bed nucleus of the stria terminalis and CeA but not in basolateral amygdala (BLA) or in the paraventricular nucleus. Injection D-Phe-CRF(12-41) in CeA but not in the BLA-blocked binge-like eating behavior. DISCUSSION These findings demonstrate that extra-hypothalamic CRF1 receptors, rather than those involved in endocrine functions, are involved in binge eating and the crucial role of CRF receptors in CeA. CRF1 receptor antagonism may represent a novel pharmacological treatment for binge-related eating disorders.

Gene expression profiling in adipose tissue of Sprague Dawley rats identifies olfactory receptor 984 as a potential obesity treatment target

The aim of the study was to identify and functionally characterize novel candidate gene/s involved in the development of resistance to diet-induced obesity in rats. In a high-fat-diet (HFD) study of rats, we found subgroups which either developed resistance to HFD-induced obesity (DR) or showed an obesity-prone phenotype (DIO). Gene expression analysis in 10 samples (5 DIO vs 5 DR) was performed. The most promising gene, OR6C3 (orthologous with rat Olr984 and mouse Olfr788) was measured by qRT-PCR in paired samples of human visceral (Vis) and subcutaneous (SC) adipose tissue (AT) (n = 225) and in sub-fractions of adipocytes and cells of stromal vascular fraction. Gene expression analyses showed Olr984 with significantly reduced mRNA expression in DR rats. In the Vis AT of human samples we found an up-regulation of OR6C3 compared to SC AT, independent of gender, glucose tolerance or type 2 diabetes. We observed significantly lower levels of SC AT OR6C3 mRNA in subjects with obesity compared to those with normal-weight or overweight. OR6C3 is more expressed in SVF than in adipocytes. Olr984 could be a novel candidate gene related to diet-induced obesity in rats. Variation in human AT mRNA expression is related to obesity parameters and glucose homeostasis and linked to the regulatory role of insulin on the Olr984.

A multidisciplinary approach to study the brain injury in Diet-Induced Obesity (DIO) rats

Obesity represents an independent risk factor for the development of cerebrovascular disease and cognitive impairment. The systemic effects, such as increased fat mass, hypertension, insulin resistance and general metabolic dysfunction, have been identified as factors that may lead to impaired cognitive function. To clarify the possible relationships between obesity and nervous system changes, high-caloric Diet-Induced Obesity (DIO) rats 7 weeks-old, were studied after 17 weeks of hypercaloric diet compared to control rats with not fat diet (Chow) or to rats not developing obesity (DIO-resistant DR). Food consumption, fat mass content, blood pressure and blood parameters were assessed. Different behavioural tests were used to estimate cognitive performance. RT-qPCR, immunochemical and immunohistochemical analysis were performed to evaluate neuronal, glial and vascular markers. The obese phenotype developd after 5 weeks of high fat diet exposure and body weight values remained higher in DIO rats compared to the control group and DR rats during the treatment. Systolic blood pressure, glycaemia and insulin were higher in DIO rats only after 17 weeks. No differences in values of total cholesterol and triglycerides were observed. Furthermore increase of thiobarbituric reactive substances and increase of oxidated proteins, was observed in the serum of DIO rats compared to Chow rats. The open-field test revealed, in the older DIO rats, a decrease of cumulative distance travelled and in the number of rearings and an increase of total immobility time. Older DIO rats only, showed a reduction of retention latency time in the passive avoidance test. RT-qPCR, immunochemical and immunohistochemical analysis showed an increased expression of the glial-fibrillary acid protein in the frontal cortex and hippocampus of older DIO rats compared to age-matched Chow and DR rats. A decrease of neurofilament expression was found in the hippocampus of older DIO rats without changes in the number of neurons. A modulation in the Transient Receptor Potential (TRP) channels and synaptic components was highlighted in cerebral areas. These results indicate that obesity in rats, in addition to the development of correlate cerebrovascular risk factors, causes brain injury characterized by astrogliosis, neurodegeneration and impaired learning and memory tasks. The identification of neurodegenerative changes in DIO rats may represent the first step to better characterize the neuronal modifications occurring in the obesity and propose pharmacological treatments or food strategies to counter them.

Metabolic effects of Tart Cherries supplementation in an animal model of obesity

Fruits and vegetables contain non-nutritive phytochemicals that may contribute to their health-promoting effects. Anthocyanins are phytochemical flavonoids principally found in fruits and vegetables. Several studies have suggested that anthocyanin-rich plant extracts can modify lipid metabolism in vitro and can reduce hyperlipidemia in vivo. Tart cherries (Prunus cerasus L.) are a rich source of anthocyanins. This study was designed to evaluate the effects of anthocyanin-rich tart cherries extract and seeds powder on Diet-Induced Obesity (DIO) rats, that provide a useful animal model sharing several common features with human obesity. DIO rats were studied for 17 weeks of hypercaloric diet with the supplementation of 0,1 mg/kg of tart cherries seeds powder (DS) and seeds powder plus tart cherries extract, containing 1mg of anthocyanins (DES). DIO rats were compared to the control rats with not fat diet (Chow).To determine the systemic effects of caloric dense expousure we examined food consumption, fat mass content and fasting glycemia, insulin levels, cholesterol and triglycerides. Ultrasonographic (US) and computed tomography (CT) evaluations were performed to detect adipose tissue deposition. In CT, also fat infarction of the liver was investigated followed by histochemical analysis 17 weeks of fat diet, rats increased significantly their body weight in comparison to the control group. Glycaemia and insulin levels were higher in DIO rats. No difference in body weight was found in DS and DES rats compared to age-matched DIO rats. Supplementation of tart cherries in DS and DES induce a decrease of the blood pressure and the glycemia. Furthermore, decreased the serum levels of thiobarbituric reactive substances. The US and CT analysis indicated an increase of deposition of visceral adipose tissue and evidenced a decrease of hepatic attenuation in DIO rats, suggests a moderate hepatic steatosis prevented by tart cherries supplementation in DS and DES rats. The evidence of the CT was confirmed by histological analysis. DIO rats present a distinctive pattern of steatosis with hepatocytic ballooning degeneration at the perivenular areas. The steatosis elements decrease in DS and DES rats. Tart cherries supplementation, although did not reduce the body weight in DIO rats, prevent the development of related risk factors. Further studies are needed to better clarify the benefits of tart cherry supplementation on health and disease prevention.

Obesity-related nervous system injury: preliminary evidences in diet induced obesity (DIO) rats

Increased food intake, reduced physical activity and altered metabolic processes are the variables that affect energy balance inducing obesity. Obesity is now considered an increasingly medical challenge. Actually, the prevalence of obesity has increased dramatically worldwide over the last decades and has now reached epidemic proportions. On the other hand, obesity is associated with the development of chronic diseases such as cerebrovascular disease promoting the cognitive decline. Caloric-dense diet induced obesity (DIO), provides a useful animal model sharing several common features with human obesity. DIO rats of 7 weeks of age are expose to high fat (45 %) diet ad libitum and after 5 weeks the obese phenotype starts to be develop. To clarify the possible relationships between obesity and nervous system changes, DIO rats were studied after 5 weeks and 17 weeks of hypercaloric diet compared to the control rats with not fat diet (Chow). Memory performance were measured using different cognitive tests. Moreover, ultrasonographic (US) and computed tomography (CT) evaluations were performed to detect adipose tissue changes. Magnetic resonance imaging (MRI) to highlight brain morphological alterations was used. Morphological changes of brain areas (frontal cortex, hippocampus) were evaluated by immunohistochemical analysis. The results confirmed the developed of obesity after 5 weeks of fat diet. At long-term (17 weeks) high fat diet exposure, rats increased significantly their body weight in comparison to the control group and the youngest DIO rats. The US and CT analysis indicated an increase of deposition of both visceral and subcutaneous adipose tissue and evidences a decrease of hepatic attenuation in the older DIO rats.MRI images did not show vascular and morphological alterations in brain. Instead, immuhistochemical and immunochemical analysis, revealed an increase expression of glial-fibrillary acidic protein (GFAP) in the older DIO rats compared to the age- matched Chow rats both in frontal cortex and in hippocampus. DIO rats showed a reduction of retention latency time in the emotional learning task. These preliminary findings indicate that the development of obesity, does not determined gross anatomy alteration in brain, but the occurrence of injury characterized by astrogliosis. The identification of neurodegenerative changes in DIO may represent the first insight to better characterize the neuronal involvement in obesity.