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Obesity is the result of genetic, behavioral, environmental, physiological, social and cultural factors that result in energy imbalance and promote excessive fat deposition. In particular, in Western society, which is characterized by sedentary lifestyles combined with excess energy intake, many people often try to lose weight with caloric restriction. However it is well known that the weight regain after caloric restriction results in accelerated storage of adipose tissue (De Andrade et al. 2015). The high efficiency of the recovery of the energy depots of body fat probably evolved in ancient times, when the food availability was intermittent and it was necessary to face up to long periods of famine. Nowadays, this efficiency is the key factor causing higher body fat gain relative to lean tissue, and the preferential catch-up fat phenomenon has also been linked to the hyperinsulinemic state of catch-up growth and the associated risks for later development of metabolic syndrome (Crescenzo et al. 2003; Dulloo et al.2006). Several studies on refeeding after caloric restriction have been conducted on laboratory rats, a very good model for obesity studies, because energy intake, diet composition and the level of physical activity can be easily monitored, and also, considering the standard housing conditions, laboratory rats exhibit a sedentary behavior, similarly to what happens in humans (Aydin et al. 2014; Buettner et al. 2007; Spangenberg et al. 2005): it was observed that during the refeeding with low fat diet the rats showed a reduction in energy expenditure and an increase in metabolic efficiency, causing high body fat deposition, even in absence of hyperphagia (Dulloo et al. 2008). In addition it has been shown that this high metabolic efficiency that drives catch-up fat on a low fat diet is exacerbated by refeeding on HFD (Crescenzo et al. 2003; Dulloo & Gerardier, 1992), although in different ways depending on the type of fat included in the diet (Dulloo et al. 2005). In this thesis, I assessed whether changing the type of fat, in the context of a high fat dietary regimen, could differently affect whole body homeostasis. For this reason I investigated the effect of two kind of HFD, rich in lard or safflower/linseed oil. I used two different experimental designs. In the first one, rats were divided in two groups with the same mean body weight and were pair fed with 380 kJ metabolisable energy (ME)/day (corresponding to the spontaneous energy intake of the same rats, as assessed in the days before the experiment) with a lard-based (SFA-MUFA, mainly monounsaturated and saturated fatty acids) or safflower-linseed based (PUFA, polyunsaturated fatty acids of ω-6 and ω-3 series) diets for 2 weeks. The two HFDs contained 58.2% energy from fat, 21.1% energy from protein and 20.7% energy from carbohydrate. During the treatments, body weight, food, and water intake were monitored daily. Faces and urine were collected daily and the respective energy content assessed with a bomb calorimeter. The day before the sacrifice, 24-h VO2 and VCO2 of the rats were recorded with a four-chamber indirect open-circuit calorimeter (Panlab S.r.l., Cornella, Spain). Measurements were performed for the whole 24 h-period every 15 min for 3 min in each cage. Urine was collected for the whole (24-h) period and urinary nitrogen levels were measured by an enzymatic colorimetric method (FAR S.r.l., Settimo di Pescantina, Italy). Daily energy expenditure and substrate oxidation rates were calculated for the whole 24-h period from VO2, VCO2, and urinary nitrogen according to Even et al. The day before the euthanasia, rats were fasted for 6 h and small blood samples were taken from the tail vein, placed in EDTA coated tubes and transferred on ice. Plasma glucose concentration was measured by colorimetric enzymatic method (Pokler Italia, Italy), while plasma insulin concentration was measured using an ELISA kit (Mercodia AB, Sweden). Basal postabsorptive values of plasma glucose and insulin were used to calculate Homeostatic Model Assessment (HOMA) index as [Glucose (mg/dL) × Insulin (mU/L)]/405. After the treatment, body composition and energy balance were measured. Plasma concentrations of triglycerides, cholesterol, non esterified fatty acids (NEFA), alanine aminotransferase (ALT) and lipid peroxidation were measured. Liver composition and oxidation, as well as respiratory capacity, oxidative status of liver mitochondria were measured. Quantification of uncoupling protein (UCP-1) in interscapular brown adipose tissue (IBAT) was performed. In the second experimental design, rats were food restricted for 14 days at approximately 50% of spontaneous intake. At the end of the semistarvation period, all the rats were separated into 3 groups (n = 8): one group was immediately euthanized by decapitation to calculate the body composition of restricted rats, while the other two groups were refed isocaloric amounts of two different HFDs (58.2% by energy), rich in lard or safflower/linseed oil. The amount of dietary energy provided to the refed animals corresponds to the metabolisable energy intake of spontaneously growing (non-restricted) weight-matched control animals fed on chow diet, as previously reported (Crescenzo et al., 2003). Furthermore, the level of fat in the HFDs utilized here (i.e., 58% of energy intake) corresponds to dietary fat levels often utilized in rehabilitation (energy-dense) diets of malnourished infants and children in order to meet their high energy requirements for catch-up growth (Prentice and Paul, 2000). In this second design, I performed the same measurement of the first one, but I also investigated the regulation of the pathway of de novo lipogenesis in liver, white adipose tissue (WAT) and brown adipose tissue (BAT). I also evaluated liver and muscle composition, mitochondrial activity, as well as parameters of oxidative stress and inflammation, since both these tissues are major contributors to daily metabolic rate (Rolfe & Brown, 1997) and it has been proposed that skeletal muscle is involved in the suppression of thermogenesis that underlines the high metabolic efficiency for accelerated body fat recovery after caloric restriction (Dulloo, 2005; Crescenzo et al. 2006). The results of the first experimental design suggest that, at the end of the treatment, the percent of body lipid doubled both in SFA-MUFA and in PUFA rats, although the final value was significantly lower in PUFA than in SFA-MUFA rats; conversely the percentage of body protein was maintained constant in PUFA rats, while it significantly decreased in SFA-MUFA rats. The percent of body epididymal and visceral WAT increased, reaching a final value that was significantly lower in PUFA rats than in SFA-MUFA rats. The percent of body IBAT was significantly higher in PUFA than in SFA-MUFA rats and its content of UCP-1 was markedly increased in PUFA rats compared to SFA-MUFA rats. PUFA rats exhibit lower lipid gain but higher protein gain compared to SFA-MUFA rats. The analysis of fuel oxidation showed that PUFA rats had reduced protein oxidation but higher lipid oxidation compared to SFA-MUFA rats. Plasma metabolic characterisation evidenced higher alanine aminotransferase activity in PUFA rats compared to SFA-MUFA rats. Livers from PUFA rats showed higher degree of steatosis and had higher lipid content, triglycerides and cholesterol, as well as higher lipid peroxidation, compared to SFA-MUFA rats. Liver mitochondria from PUFA rats displayed a significant decrease in basal and fatty acid-induced proton leak compared to SFA-MUFA rats. Both NAD, FAD and lipid-linked maximal oxidative capacities were significantly higher in isolated liver mitochondria from rats PUFA compared to SFA-MUFA rats. Evaluation of oxidative status showed that lipid peroxidation was significantly higher in mitochondria from PUFA rats compared to SFA-MUFA rats. The results of the second experimental design suggest that rats refed the PUFA-enriched diet had lower body lipids and higher body proteins compared to rats refed the SFA-MUFA-enriched diet, as well as lower amount of visceral and epididymal WAT, but higher amount of IBAT. Rats refed PUFA diet gained significantly less lipids and more proteins. At the end of the 2 weeks refeeding period, rats refed PUFA diet exhibited higher NPRQ values and non-protein energy utilisation was fulfilled by using proportionally more carbohydrates and less fat compared to rats refed SFA-MUFA diet, lower HOMA index and plasma insulin levels in the fasting and the fed state, together with lower plasma triglycerides and cholesterol levels. Plasma lipid peroxidation was not significantly different between the two groups of rats, while a significant decrease in ALT was found in rat refed PUFA diet. FAS activity, the rate-limiting enzyme in the pathway of de novo lipogenesis, was found to be significantly higher in liver, e-WAT and IBAT in rat refed PUFA diet. In the liver, higher triglyceride content was found in rat refed PUFA diet, but hepatic lipid peroxidation was significantly lower. SOD activity was found to be significantly higher in liver mitochondria of rat refed PUFA diet while no difference was found in skeletal muscle mitochondria. Lower degree of hepatic inflammation and unchanged hepatic content of the proinflammatory mediator TNF-α was found in rat refed PUFA diet. Expression of the UCP-1 protein in BAT was found significantly increased in rat refed PUFA diet. In conclusion, in this thesis I provide evidence that not only the amount, but also the type of dietary fat, is a primary obesogenic factor. In particular, when considering the composition of high fat diets for nutritional rehabilitation, the inclusion of PUFA could be useful for improving protein deposition and maintaining glucose homeostasis, while limiting lipid storage in adipose tissue and oxidative stress and inflammation in the liver.

The increase in the use of refined food, which is rich in fructose, is of particular concern in children and adolescents, since the total caloric intake and the prevalence of metabolic syndrome are increasing continuously in these populations. Nevertheless, the effects of high fructose diet have been mostly investigated in adults, by focusing on the effect of a long-term fructose intake. Notably, some reports evidenced that even short-term fructose intake exerts detrimental effects on metabolism. Therefore, the aim of this study was to compare the metabolic changes induced by the fructose-rich diet in rats of different age, i.e., young (30 days old) and adult (90 days old) rats. The fructose-rich diet increased whole body lipid content in adult, but not in young rats. The analysis of liver markers of inflammation suggests that different mechanisms depending on the age might be activated after the fructose-rich diet. In fact, a pro-inflammatory gene-expression analysis showed just a minor activation of macrophages in young rats compared to adult rats, while other markers of low-grade metabolic inflammation (TNF-alpha, myeloperoxidase, lipocalin, haptoglobin) significantly increased. Inflammation was associated with oxidative damage to hepatic lipids in young and adult rats, while increased levels of hepatic nitrotyrosine and ceramides were detected only in young rats. Interestingly, fructose-induced hepatic insulin resistance was evident in young but not in adult rats, while whole body insulin sensitivity decreased both in fructose-fed young and adult rats. Taken together, the present data indicate that young rats do not increase their body lipids but are exposed to metabolic perturbations, such as hepatic insulin resistance and hepatic oxidative stress, in line with the finding that increased fructose intake may be an important predictor of metabolic risk in young people, independently of weight status. These results indicate the need of corrective nutritional interventions for young people and adults as well for the prevention of fructose-induced metabolic alterations.

The recovery of body weight after a period of caloric restriction is accompanied by an enhanced efficiency of fat deposition and hyperinsulinemia—which are exacerbated by isocaloric refeeding on a high fat diet rich in saturated and monounsaturated fatty acids (SFA-MUFA), and poor in polyunsaturated fatty acids (PUFA), and associated with a blunting of de novo lipogenesis in adipose tissue and liver. As high fat diets rich in PUFA have been shown to limit the excess fat deposition and improve glucose homeostasis, we investigated here the extent to which de novo lipogenesis in liver and adipose tissues (white and brown), as well as hepatic oxidative stress, are influenced by refeeding on diets rich in PUFA.Design: In rats calorically restricted for 14 days and refed for 14 days on isocaloric amounts of a high fat diet rich in lard (i.e., high SFA- MUFA) or in safflower and linseed oils (rich in PUFA), we investigated energy balance, body composition, glycemic profile, and the regulation of fatty acid synthase (rate- limiting enzyme of de novo lipogenesis) in liver, white and brown adipose tissue. We also evaluated oxidative stress in liver and skeletal muscle and markers of hepatic inflammation.Results: Rats refed the PUFA diet gained less lipids and more proteins compared to rats refed SFA-MUFA diet and showed lower amount of visceral and epididymal white adipose tissue, but increased depots of interscapular brown adipose tissue, with higher expression of the uncoupling protein 1. A significant increase in non- protein respiratory quotient and carbohydrate utilization was found in rats refed PUFA diet. Rats refed PUFA diet showed improved glucose homeostasis, as well as lower triglycerides and cholesterol levels. Fatty acid synthase activity was significantly higher in liver, white and brown adipose tissue, while lipid peroxidation and the degree of inflammation in the liver were significantly lower, in rats refed PUFA diet.Conclusions: When considering the composition of high fat diets for nutritional rehabilitation, the inclusion of PUFA could be useful for improving protein deposition and maintaining glucose homeostasis, while limiting lipid storage in adipose tissue and oxidative stress and inflammation in the liver.