Leptin Expression in Hypothalamic PVN Reverses Dietary Obesity and Hyperinsulinemia but Stimulates Ghrelin*

Objective: In order to circumvent the multiple peripheral effects of hyperleptinemia and leptin resistance, the efficacy of leptin transgene expression in the hypothalamic paraventricular nucleus (PVN) to reinstate the central energy homeostasis in obesity was examined. Research Methods and Procedures: A recombinant adeno‐associated viral vector encoding either leptin (rAAV‐lep) or green fluorescent protein (rAAV‐GFP) was microinjected into the PVN of obesity‐prone rats consuming a high‐fat diet (HFD). Results: rAAV‐lep, and not rAAV‐GFP, microinjection significantly reduced energy intake and enhanced energy expenditure, thereby resulting in normalization of weight and blood levels of leptin, insulin, free fatty acids, and glucose concomitant with enhanced ghrelin secretion during the extended period of observation. Discussion: Thus, we show, for the first time, that amelioration of leptin insufficiency with enhanced localized leptin availability in the PVN alone can reverse dietary obesity and the attendant hyperinsulinemia and concurrently block the central stimulatory effects of elevated endogenous ghrelin on food intake and adiposity.     

Viral mediated neuropeptide Y expression in the rat paraventricular nucleus results in obesity

Objective: Chronic central administration of neuropeptide Y (NPY) has dramatic effects on energy balance; however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of NPY signaling in the PVN to energy balance. Research Methods and Procedures: Recombinant adeno‐associated viral particles containing NPY (rAAV‐NPY) were injected in the rat brain with coordinates targeted at the PVN. For three weeks, body weight, food intake, endocrine parameters, body temperature, and locomotor activity were measured. Furthermore, effects on insulin sensitivity and expression of NPY, agouti‐related protein (AgRP), and pro‐opiomelanocortin in the arcuate nucleus were studied. Results: Food intake was increased specifically in the light period, and dark phase body temperature and locomotor activity were reduced. This resulted in obesity characterized by increased fat mass; elevated plasma insulin, leptin, and adiponectin; decreased AgRP expression in the arcuate nucleus; and decreased insulin sensitivity; whereas plasma corticosterone was unaffected. Discussion: These data suggest that increased NPY expression targeted at the PVN is sufficient to induce obesity. Interestingly, plasma concentrations of leptin and insulin were elevated before a rise in food intake, which suggests that NPY in the PVN influences leptin and insulin secretion independently from food intake. This strengthens the role of the PVN in regulation of energy balance by NPY.     

Sustained NPY Overexpression in the PVN Results in Obesity via Temporarily Increasing Food Intake

Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno‐associated virus (rAAV)‐mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair‐fed a group of AAV‐NPY‐injected rats to AAV‐control‐injected rats and compared parameters of energy balance to ad libitum fed AAV‐NPY‐injected rats. For 3 weeks, AAV‐NPY‐injected rats, received the same amount of food as ad libitum‐fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV‐NPY‐injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV‐NPY‐injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.     

Altered Hypothalamic Signaling and Responses to Food Deprivation in Rats Fed a Low‐Carbohydrate Diet

Objective: To model how consuming a low‐carbohydrate (LC) diet influences food intake and body weight. Research Methods and Procedures: Food intake and body weight were monitored in rats with access to chow (CH), LC‐high‐fat (HF), or HF diets. After 8 weeks, rats received intracerebroventricular injections of a melanocortin agonist (melanotan‐II) and antagonist (SHU9119), and feeding responses were measured. At sacrifice, plasma hormones and hypothalamic expression of mRNA for proopiomelanocortin (POMC), melanocortin‐4 receptor, neuropeptide Y (NPY), and agouti related protein (AgRP) were assessed. A second set of rats had access to diet (chow or LC‐HF) for 4 weeks followed by 24 h food deprivation on two occasions, after which food intake and hypothalamic POMC, NPY, and AgRP mRNA expression were measured. Results: HF rats consumed more food and gained more weight than rats on CH or LC‐HF diets. Despite similar intakes and weight gains, LC‐HF rats had increased adiposity relative to CH rats. LC‐HF rats were more sensitive to melanotan‐II and less sensitive to SHU9119. LC‐HF rats had increased plasma leptin and ghrelin levels and decreased insulin levels, and patterns of NPY and POMC mRNA expression were consistent with those of food‐deprived rats. LC‐HF rats did not show rebound hyperphagia after food deprivation, and levels NPY, POMC, and AgRP mRNA expression were not affected by deprivation. Discussion: Our results demonstrate that an LC diet influences multiple systems involved in the controls of food intake and body weight. These data also suggest that maintenance on an LC‐HF diet affects food intake by reducing compensatory responses to food deprivation.     

Paraventricular opioids alter intake of high-fat but not high-sucrose diet depending on diet preference in a binge model of feeding

Previous work from our laboratory indicates that when rats are given a choice between a high-fat and a high-sucrose diet, opioid blockade with naltrexone (NTX) in a reward-related site (central amygdala) inhibits intake of the preferred diet only, whereas NTX injected into a homeostasis-related site, such as the hypothalamic paraventricular nucleus (PVN), inhibits intake of both diets. However, other work suggests that opioids increase intake of fat specifically. The present study further investigates the role of PVN opioids in food choices made by calorically-replete animals. We used a binge model with chow-maintained rats given 3-h access to a choice of a high-fat or high-sucrose diet 3 days a week. We hypothesized that intra-PVN injection of the mu-opioid agonist, DAMGO (0, 0.025, 0.25, and 2.5 nmol) would enhance, and NTX (0, 10, 30, and 100 nmol) would inhibit intake of both diets to an equal extent. We found that when animals were divided into groups according to sucrose or fat preference, DAMGO increased fat intake in fat-consuming animals, while having no effect on intake of either diet in sucrose-consuming animals. NTX, however, inhibited fat intake in both groups. Intra-PVN NTX did not inhibit intake of sucrose when presented in the absence of a fat choice, but did so when injected peripherally. Furthermore, intra-PVN and systemic NTX inhibited intake of chow by 24-h-food-deprived animals. These results indicate a complex role for PVN opioids in food intake with preference, nutrient type, and energy state affecting the ability of these compounds to change behavior.     

Intake of trans fatty acids during gestation and lactation leads to hypothalamic inflammation via TLR4/NFκBp65 signaling in adult offspring

We examined whether feeding pregnant and lactating rats with hydrogenated vegetable fats rich in trans fatty acids led to an increase in serum endotoxin levels and inflammation and to impaired satiety-sensing pathways in the hypothalamus of 90-day-old offspring. Pregnant and lactating Wistar rats were fed either a standard chow (Control) or one enriched with hydrogenated vegetable fat (Trans). Upon weaning, the male offspring were divided in two groups: Control-Control (CC), mothers and offspring fed the control diet; and Trans-Control (TC), mothers fed the trans diet, and offspring fed the control diet. The offspring's food intake and body weight were quantified weekly and the offspring were killed on the 90th day of life by decapitation. The blood and hypothalamus were collected from the offspring. Food intake and body weight were higher in the TC rats than in the CC rats. TC rats had increased serum endotoxin levels and increased hypothalamic cytokines, IL-6, TNF-α and IL1-β, concentrations (P<.05). TLR4, NFκBp65 and MyD88 were higher (P<.05) in the TC rats than in the CC rats. AdipoR1 was lower in the TC rats than in the CC rats. Thus, the present study shows that the mothers' hydrogenated vegetable fat intake during pregnancy and lactation led to hypothalamic inflammation and impaired satiety-sensing, which promotes deleterious metabolic consequences such as obesity, even after the withdrawal of the causal factor. In other words, the effect remains after the consumption of the standard chow by offspring.     

High-fat diet offsets the long-lasting effects of running-wheel access on food intake and body weight in OLETF rats

We have previously demonstrated that running-wheel access normalizes the food intake and body weight of Otsuka Long-Evens Tokushima Fatty (OLETF) rats. Following 6 wk of running-wheel access beginning at 8 wk of age, the body weight of OLETF rats remains reduced, demonstrating a lasting effect on their phenotype. In contrast, access to a high-fat diet exacerbates the hyperphagia and obesity of OLETF rats. To determine whether diet modulates the long-term effects of exercise, we examined the effects of high-fat diet on food intake and body weight in OLETF rats that had prior access to running wheels for 4 wk. We found that 4 wk of running exercise significantly decreased food intake and body weight of OLETF rats. Consistent with prior results, 4 wk of exercise also produced long-lasting effects on food intake and body weight in OLETF rats fed a regular chow. When running wheels were relocked, OLETF rats stabilized at lower levels of body weight than sedentary OLETF rats. However, access to a high-fat diet offset these effects. When OLETF rats were switched to a high-fat diet following wheel relocking, they significantly increased food intake and body weight, so that they reached levels similar to those of sedentary OLETF rats fed a high-fat diet. Gene expression determination of hypothalamic neuropeptides revealed changes that appeared to be appropriate responses to the effects of diet and running exercise. Together, these results demonstrate that high-fat diet modulates the long-lasting effects of exercise on food intake and body weight in OLETF rats.     

Bingeing,Self‐restriction,and Increased Body Weight in Rats With Limited Access to a Sweet‐fat Diet

Objective: Prior research has shown that fasting alternated with a diet of standard rodent chow and a 10% sucrose solution produces bingeing on the sucrose, but animals remain at normal body weight. The present study investigated whether restricted access to a highly palatable combination of sugar and fat, without food deprivation, would instigate binge eating and also increase body weight. Methods and Procedures: Male rats were maintained for 25 days on one of four diets: (i) sweet‐fat chow for 2 h/day followed by ad libitum standard chow, (ii) 2‐h sweet‐fat chow only 3 days/week and access to standard chow the rest of the time, (iii) ad libitum sweet‐fat chow, or (iv) ad libitum standard chow. Results: Both groups with 2‐h access to the sweet‐fat chow exhibited bingeing behavior, as defined by excessively large meals. The body weight of these animals increased due to large meals and then decreased between binges as a result of self‐restricted intake of standard chow following binges. However, despite these fluctuations in body weight, the group with 2‐h access to sweet‐fat chow every day gained significantly more weight than the control group with standard chow available ad libitum. Discussion: These findings may have implications for the body weight fluctuations associated with binge‐eating disorder, as well as the relationship between binge eating and the obesity epidemic.     

Influence of diet form on the hyperphagia-promoting effect of polysaccharide in rats

Adult female rats were fed chow only, or chow plus the polysaccharide Polycose presented as a solution (32% w/v), as a powder, or as a powder mixed into the chow diet. The rats fed the Polycose solution overate and gained three times as much weight as did the control rats in the 30-day test period. The rats fed the Polycose powder or mixed diet, on the other hand, did not differ from controls in food intake or weight gain. The solution-fed rats consumed more Polycose than did the powder-fed rats, but the two groups were equivalent in their chow intake. The absolute and percent body fat of the solution-fed rats exceeded that of control rats, as well as that of powder and mixed-diet animals. The percent body fat of the powder and mixed diet groups exceeded that of the control animals. The findings demonstrate that the form of diet presentation has a major impact on the rat's feeding and body weight responses to polysaccharide diets.     

Regulation of hypothalamic NPY by diet and smoking

Appetite is regulated by a number of hypothalamic neuropeptides including neuropeptide Y (NPY), a powerful feeding stimulator that responds to feeding status, and drugs such as nicotine and cannabis. There is debate regarding the extent of the influence of obesity on hypothalamic NPY. We measured hypothalamic NPY in male Sprague-Dawley rats after short or long term exposure to cafeteria-style high fat diet (32% energy as fat) or laboratory chow (12% fat). Caloric intake and body weight were increased in the high fat diet group, and brown fat and white fat masses were significantly increased after 2 weeks. Hypothalamic NPY concentration was only significantly decreased after long term consumption of the high fat diet. Nicotine decreases food intake and body weight, with conflicting effects on hypothalamic NPY reported. Body weight, plasma hormones and brain NPY were investigated in male Balb/c mice exposed to cigarette smoke for 4 days, 4 and 12 weeks. Food intake was significantly decreased by smoke exposure (2.32+/-0.03g/24h versus 2.71+/-0.04g/24h in control mice (non-smoke exposed) at 12 weeks). Relative to control mice, smoke exposure led to greater weight loss, while pair-feeding the equivalent amount of chow caused an intermediate weight loss. Chronic smoke exposure, but not pair-feeding, was associated with decreased hypothalamic NPY concentration, suggesting an inhibitory effect of cigarette smoking on brain NPY levels. Thus, consumption of a high fat diet and smoke exposure reprogram hypothalamic NPY. Reduced NPY may contribute to the anorexic effect of smoke exposure.     

Comparison of Fat Storage between Fischer 344 and Obesity‐Resistant Lou/C Rats Fed Different Diets**

Objective: We aimed to characterize further the Lou/C (LOU) and Fischer 344 (F344) rat strains for nutritional traits to validate their use as contrasting strains for molecular genetic studies. Research Methods and Procedures: Five batches of LOU and F344 rats were used to measure caloric intake, weight gain, and body composition when fed a chow diet, a self‐selection diet (together with the study of preferences for macronutrients), hypercaloric diets, and a chow diet in a cold environment. Results: Despite a higher caloric intake when fed a chow diet, LOU rats showed a lower weight gain, final body weight, and percentage of fat tissue, together with a higher percentage of carcass weight, than F344 rats. When fed a self‐selection diet, LOU males ingested less protein and more fat than F344 males, and the reverse was observed for females. In this condition, feed efficiency was reduced in LOU but increased in F344 rats compared with the chow diet. Diet‐induced obesity was observed in F344 rats but not in LOU rats fed hypercaloric diets. In a cold environment, both LOU and F344 rats displayed an increased percentage of brown adipose tissue compared with control groups, together with a higher caloric intake. Discussion: The study shows robust nutritional differences between the LOU rat, a lean strain with a low feed efficiency and resistant to diet‐induced obesity, and the contrasting F344 rat strain. It also shows the interest in these strains for studying the genetic components of resistance to obesity.     

Mice with Low Metabolic Rates Are Not Susceptible to Weight Gain When Fed a High‐Fat Diet

Objective: Mice divergently selected for high or low food intake (FI) at constant body mass differ in their resting metabolic rates (RMRs). Low‐intake individuals (ML) have significantly lower RMR (by 30%) compared with those from the high‐intake line (MH). We hypothesized that MLs might, therefore, be more likely to increase their body and fat mass when exposed to a high‐fat diet (HFD). Research Methods and Procedures: We exposed both lines to a diet with 44.9% calories from fat for 3 weeks while measuring FI, fecal production, and body mass and then returned the mice to standard chow. Results: When exposed to the HFD, both lines significantly decreased their FI (MH, 40% to 45%; ML, 31% to 35%). This decrease occurred simultaneously with a significant increase in apparent energy absorption efficiency (AEAE). When returned to chow, FI and AEAE returned to the levels observed prior to HFD exposure. Because of the adjustments in FI, the absorbed energy was maintained in the MLs and, thus, body mass remained constant. The MH individuals overcompensated for the elevated energy content and AEAE on the HFD and, therefore, absorbed lower energy than when feeding on chow. These mice also did not significantly change their body mass when on the HFD and must have made adjustments in their energy expenditures. Both lines and both sexes increased in fat content on the HFD, but these effects were not different between lines or sexes. Discussion: We found no support for the hypothesis that mice with low RMRs were more susceptible to weight gain when fed the HFD.     

Normal Distribution of Body Weight Gain in Male Sprague‐Dawley Rats Fed a High‐Energy Diet

Objective: To investigate the effect of a high‐energy (HE) diet on caloric intake, body weight, and related parameters in outbred male Sprague‐Dawley (SD) rats. Research Methods and Procedures: Twenty‐eight SD rats were fed either chow (C) for 19 weeks or HE diet for 14 weeks and then C for 5 weeks. Blood hormones and metabolites were assayed, and expression of uncoupling protein‐1 and hypothalamic energy‐balance‐related genes were determined by Northern blotting and in situ hybridization, respectively. Results: HE rats gained body weight more rapidly than C animals with a range of weight gains, but there was no evidence that weight gain was bimodally distributed. Caloric intake was transiently elevated after introduction of the HE diet. Transfer of HE rats back to C resulted in a drop in caloric intake, but a stable body weight. In terminal analysis, two of four dissected adipose tissue depots were heavier in rats that had previously been fed HE diet. Blood leptin, insulin, glucose, and nonesterified fatty acids were not different between the groups. Uncoupling protein‐1 mRNA was elevated in interscapular brown adipose tissue from HE rats. There was a trend for agouti‐related peptide mRNA in the hypothalamic arcuate nucleus to be higher in HE rats. Discussion: Contrary to other studies of the SD rat on HE diet, body weight and other measured parameters were normally distributed. There was no segregation into two distinct populations on the basis of susceptibility to diet‐induced obesity. This characteristic may be dependent on the breeding colony from which animals were sourced.     

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β₃ adrenergic receptor (β₃‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β₃‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.     

Intentional Weight Loss Reduces Mortality Rate in a Rodent Model of Dietary Obesity

Objective: We used a rodent model of dietary obesity to evaluate effects of caloric restriction‐induced weight loss on mortality rate. Research Measures and Procedures: In a randomized parallel‐groups design, 312 outbred Sprague‐Dawley rats (one‐half males) were assigned at age 10 weeks to one of three diets: low fat (LF; 18.7% calories as fat) with caloric intake adjusted to maintain body weight 10% below that for ad libitum (AL)‐fed rat food, high fat (HF; 45% calories as fat) fed at the same level, or HF fed AL. At age 46 weeks, the lightest one‐third of the AL group was discarded to ensure a more obese group; the remaining animals were randomly assigned to one of three diets: HF‐AL, HF with energy restricted to produce body weights of animals restricted on the HF diet throughout life, or LF with energy restricted to produce the body weights of animals restricted on the LF diet throughout life. Life span, body weight, and leptin levels were measured. Results: Animals restricted throughout life lived the longest (p < 0.001). Life span was not different among animals that had been obese and then lost weight and animals that had been nonobese throughout life (p = 0.18). Animals that were obese and lost weight lived substantially longer than animals that remained obese throughout life (p = 0.002). Diet composition had no effect on life span (p = 0.52). Discussion: Weight loss after the onset of obesity during adulthood leads to a substantial increase in longevity in rats.