Adolescent activity-based anorexia has a substantial and prolonged impact on social behavior in young adult female rats.

Activity-based anorexia (ABA) is a rodent model of anorexia nervosa (AN) that induces several key components of AN, including voluntary reduction in food intake, reduced body weight, hyperactivity, and alterations to the hypothalamic-pituitary-adrenal (HPA) axis. Previous research has demonstrated persistently increased anxiety-like behavior in the elevated plus maze (EPM), a test measuring avoidance of novel and open areas in adult female rats that experienced ABA during adolescence and are weight-restored in adulthood. Whether the same behavioral effects of two bouts of adolescent ABA emerge in response to different anxiety-provoking stimuli, however, has not been explored. We used the social partition (SP), novelty suppressed feeding (NSF), marble burying, and EPM tests to explore whether two bouts of adolescent ABA have persistent effects on anxiety-like behavior in weight restored young adult female rats. One-way ANOVA analyses revealed that female rats that experienced two bouts of ABA during adolescence had increased anxiety-like behavior in the EPM and SP tests in young adulthood following weight restoration compared with controls. These data demonstrate that the enduring behavioral effects of two bouts of adolescent ABA are specific to particular anxiety-provoking stimuli and suggest that adolescent ABA has enduring effects on social relationships.

Fear extinction is impaired in aged rats.

Normal aging is accompanied by broad loss of cognitive function in humans and rodents, including declines in cognitive flexibility. In extinction, a conditional stimulus (CS) that was previously paired with a footshock is presented alone. This procedure reliably reduces conditional freezing behavior in young adult rats. Here, we aimed to investigate how normal aging affects extinction learning. Using young (3 months) and aged (20 months) male and female Long Evans rats, we compared extinction (using 20 CS-alone presentations) to a no extinction control (equal exposure to the conditioning chamber without CS presentations) following delay fear conditioning. We found that young animals in the extinction group showed a decrease in freezing following extinction; aged animals did not. We next examined changes in neural activity using expression of the immediate early gene zif268. In young animals, extinction corresponded with decreased expression of zif268 in the basolateral amygdala and anterior retrosplenial cortex; this was not observed in aged animals. Further, aged animals showed increased zif268 expression in each region examined, suggesting that dysfunction in neural activity precedes cognitive deficits. These results demonstrate that aging impacts both extinction learning and neural activity.

Sex-specific effects of ketogenic diet after pre-exposure to a high-fat, high-sugar diet in rats.

BACKGROUND AND AIMS: The objectives were to evaluate the relationship between ketogenic diets, the ketone body beta-hydroxybutyrate (BHB), parameters known to increase risk for cardiovascular and metabolic diseases in both sexes, using a pre-clinical model of obesity. METHODS AND RESULTS: Rats had access to a diet high in fat and sugar (HFS) for 12 weeks. After HFS, they switched to chow (HFS-CH) or ketogenic diet (HFS-KD) for 3 weeks to model a dietary intervention. Body weight, adiposity, and food intake were measured. Glucose tolerance and corticosterone response to stress were measured after HFS, then again after the intervention. Both sexes increased body weight, food intake, and adiposity compared to control (CTL) while on HFS. HFS females showed impaired glucose tolerance. HFS males developed a dampened corticosterone to stress, whereas HFS females developed an exacerbated response. The effects of HFS on adiposity and corticosterone were reversed in HFS-CH males. These same improvements were observed in HFS-CH females, although they still had impaired glucose tolerance. HFS-KD males showed some improvements, however, they still had higher body weight and adiposity than CTL. The same pattern was observed in females. These beneficial effects of KD correlated with plasma BHB levels in females but not in males. CONCLUSIONS: These data model effects reported in clinical literature and serve as a valuable translational tool to further test causal mechanisms that lead to desirable outcomes of KD. These sex-specific relationships are important, as KD could potentially affect endocrine mechanisms differently in males and females.

Dual functions of CNS inflammation in food intake and metabolic regulation.

Western diet (WD) consumption induces chronic mild inflammation in the hypothalamus. However, metabolic consequences of increased hypothalamic inflammatory cytokines remain unclear. This research first aimed to examine whether increased proinflammatory cytokines in the brain influenced feeding or metabolism. Rats that received an intracerebroventricular third ventricle injection (i3vt) of 0.5 pg TNFα daily for six days consumed significantly more calories than saline-injected rats, with no differences between treatment groups in terms of body weight, blood triglycerides nor glucose regulation. Continuously infusing TNFα for three weeks decreased hepatic fatty acid synthase (FAS) and increased body weight and the epididymal adipose sterol regulatory element-binding protein 1c (SREBP-1c) gene expression. Differences were not due to food intake nor voluntary wheel running activity. The second aim of this research was to examine whether inhibition of inflammation signaling in the brain at early stage of switching from chow to WD would affect diet-induced obesity development. WD-fed rats with i3vt NFκB inhibitor had greater caloric intake than rats given i3vt saline. These studies suggest elevated inflammatory cytokines in the brain induce food intake acutely and favor fat storage and weight gain in the long term. However, in the early stage of WD consumption, hypothalamic inflammatory signaling inhibits caloric intake and may serve as a warning signal of energy imbalance.

Attenuation of stress-induced weight loss with a ketogenic diet.

Ketogenic diets (KDs) are high-fat, low-carbohydrate diets that have been used therapeutically for decades, most notably for the treatment of epilepsy and diabetes. Recent data, however, suggest that KD may impart protective effects on mood disorders. The current experiments test the hypothesis that KDs can protect from stress-induced symptoms of mood disorders. To test this, we assessed behavioral and neuroendocrine effects of KD in male and female Long Evans rats. Animals experienced three weeks of chronic mild stress (CMS) while consuming KD or control chow (CH). Body weight and food intake data were recorded daily and behaviors were assayed after three weeks. Plasma beta-hydroxybutyrate (ßHB), corticosterone (CORT) and interleukin-1 beta (IL-1ß) were measured after behavioral testing, along with hypothalamic corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY) mRNA expression. CMS induced weight loss in the CH groups, however the KD-fed rats were resistant to CMS-induced weight loss. Female rats fed KD were protected from CMS-induced reductions in plasma CORT and hypothalamic NPY expression. Collectively, these data suggest protective potential of KDs against chronic stress, particularly in females.

Dietary Slowly Digestible Starch Triggers the Gut-Brain Axis in Obese Rats with Accompanied Reduced Food Intake.

SCOPE: Slowly digestible starch (SDS), as a functional carbohydrate providing a slow and sustained glucose release, may be able to modulate food intake through activation of the gut-brain axis. METHODS AND RESULTS: Diet-induced obese rats were used to test the effect on feeding behavior of high-fat (HF) diets containing an SDS, fabricated to digest into the ileum, as compared to rapidly digestible starch (RDS). Ingestion of the HF-SDS diet over an 11-week period reduced daily food intake, through smaller meal size, to the same level as a lean body control group, while the group consuming the HF-RDS diet remained at a high food intake. Expression levels (mRNA) of the hypothalamic orexigenic neuropeptide Y (NPY) and Agouti-related peptide (AgRP) were significantly reduced, and the anorexigenic corticotropin-releasing hormone (CRH) was increased, in the HF-SDS fed group compared to the HF-RDS group, and to the level of the lean control group. CONCLUSION: SDS with digestion into the ileum reduced daily food intake and paralleled suppressed expression of appetite-stimulating neuropeptide genes associated with the gut-brain axis. This novel finding suggests further exploration involving a clinical study and potential development of SDS-based functional foods as an approach to obesity control.

Reprint of "Repeated adolescent activity-based anorexia influences central estrogen signaling and adulthood anxiety-like behaviors in rats".

Anorexia nervosa (AN) typically presents in adolescence and is highly comorbid with anxiety and depression, which often persist after elimination of AN symptomology. The activity-based anorexia (ABA) paradigm allows for evaluation of behavioral and neuroendocrine consequences of AN-like behaviors, including voluntary anorexia, hyperactivity, and disruption of the hypothalamic-pituitary-gonadal (HPG) and the hypothalamic pituitary adrenal (HPA) axis. Because ABA in adolescent females results in increased anxiety-like behavior in adulthood and the estrogen signaling system has been shown to play a role in anxiety and food intake, we investigated the role of ovarian hormones in adolescent ABA-treated rats, and long-term effects of mid- and late adolescent ABA exposure on behavior and estrogen signaling. While previous research demonstrated that two bouts of ABA during adolescence resulted in decreased time in the open arm of the elevated plus maze (EPM) and increased activity of the HPA axis in response to a novel stressor, here we show that one bout of ABA in mid-or late-adolescence did not result in the same behavioral outcome. Two exposures to ABA during adolescence were necessary to produce long-term anxiety-like behavior on the EPM. Finally, removal of ovarian hormones by ovariectomy (OVX) prior to puberty did not attenuate long-term behavioral consequences of ABA in adolescence, and estrogen receptor ß (ERß) expression level in the amygdala of ABA rats was significantly lower than control subjects. Taken together, these studies identify enduring effects of ABA in adolescent females that may be mediated by ABA-induced changes to CNS ERß signaling that increase anxiety-like behaviors.

Western diets induce blood-brain barrier leakage and alter spatial strategies in rats.

Western diet (WD) intake induces obesity and metabolic dysfunction. The present study examined the effects of WD on hippocampal-dependent cognitive functioning and blood-brain barrier (BBB) permeability as a function of exposure duration, obesity phenotype, and peripheral markers of energy regulation. The use of hippocampal-dependent "place" or hippocampal-independent "response" strategies in a Y maze was assessed in male rats following 10, 40, and 90 days of WD exposure in diet-induced obese (DIO) rats, in diet resistant (DR) rats that are relatively insensitive to the obesogenic properties of WD, and in chow-fed controls. Insulin, glucose, and BBB permeability throughout several loci in the hippocampus, striatum, and cerebellum were evaluated in relation to duration of WD exposure, obesity phenotype, and type of strategy used. DIO rats had increased body weight and adiposity throughout the study, and elevated 10-day glucose and 90-day insulin levels. Throughout the study, chow-fed and DR rats reliably relied on a place strategy. DIO rats, in contrast, favored a response strategy at the 10- and 90-day time points. BBB leakage was observed in the dorsal striatum and multiple subregions of the hippocampus of DIO, but not DR or chow-fed rats. Increased ventral hippocampal BBB permeability and blood glucose levels were associated with reduced place strategy use. These data indicate that WD-induced BBB leakage is dependent on duration of diet exposure as well as obesity phenotype, and implicates BBB leakage and impaired glucoregulation in behavioral strategy and cognitive performance.

Brain and behavioral perturbations in rats following Western diet access.

Energy dense "Western" diets (WD) are known to cause obesity as well as learning and memory impairments, blood-brain barrier damage, and psychological disturbances. Impaired glucose (GLUT1) and monocarboxylate (MCT1) transport may play a role in diet-induced dementia development. In contrast, ketogenic diets (KD) have been shown to be neuroprotective. We assessed the effect of 10, 40 and 90 days WD, KD and Chow maintenance on spontaneous alternation (SA) and vicarious trial and error (VTE) behaviors in male rats, then analyzed blood glucose, insulin, and ketone levels; and hippocampal GLUT1 and MCT1 mRNA. Compared to Chow and KD, rats fed WD had increased 90 day insulin levels. SA was decreased in WD rats at 10, but not 40 or 90 days. VTE was perturbed in WD-fed rats, particularly at 10 and 90 days, indicating hippocampal deficits. WD rats had lower hippocampal GLUT1 and MCT1 expression compared to Chow and KD, and KD rats had increased 90 day MCT1 expression compared to Chow and WD. These data suggest that WD reduces glucose and monocarboxylate transport at the hippocampus, which may result in learning and memory deficits. Further, KD consumption may be useful for MCT1 transporter recovery, which may benefit cognition.

Diet-induced obesity and insulin resistance spur tumor growth and cancer cachexia in rats bearing the Yoshida sarcoma.

Obesity and insulin resistance are associated with increased risk of cancer and cancer mortality. However, it is currently unknown whether they contribute to the development of cancer cachexia, a syndrome that contributes significantly to morbidity and mortality in individuals with cancer. The present experiment addresses the question of whether preexisting obesity and insulin resistance alter tumor growth and cancer cachexia symptoms in Yoshida sarcoma bearing male rats. Obesity and insulin resistance were induced through 5 weeks of high-fat (HF) diet feeding and insulin resistance was confirmed by intraperitoneal glucose tolerance testing. Chow-fed animals were used as a control group. Following the establishment of insulin resistance, HF- and chow-fed animals were implanted with fragments of the Yoshida sarcoma or received a sham surgery. Tumor growth rate was greater in HF-fed animals, resulting in larger tumors. In addition, cancer cachexia symptoms developed in HF-fed animals but not chow-fed animals during the 18-day experiment. These results support a stimulatory effect of obesity and insulin resistance on tumor growth and cancer cachexia development in Yoshida sarcoma-bearing rats. Future research should investigate the relationship between obesity, insulin resistance, and cancer cachexia in human subjects.

Chronic exendin-4 treatment prevents the development of cancer cachexia symptoms in male rats bearing the Yoshida sarcoma.

Cancer cachexia is the syndrome of weight loss, loss of appetite, and wasting of skeletal muscle and adipose tissue experienced by many individuals with cancer. Currently, few effective treatment and prevention strategies are available for these patients, due in part to a poor understanding of the mechanisms contributing to cachexia. Insulin resistance has been associated with cancer cachexia in epidemiological, human, and animal research. The present experiment was designed to examine the ability of Exendin-4, a GLP-1 agonist and insulin sensitizing agent, to prevent the development of cachexia symptoms in male Sprague Dawley rats bearing the Yoshida sarcoma. Following tumor implantation or sham surgery, rats were treated daily with saline or Exendin-4 (3 µg/kg body weight/day) and were monitored for tumor growth and cachexia symptoms for 21-23 days. As a result of large variability in treatment effects, data were analyzed separately for animals with large and small tumors. Exendin-4 treatment reduced tumor growth and prevented the development of cancer cachexia symptoms in animals with small, but not large, tumors. In addition, insulin levels were preserved in Exendin-4-treated tumor-bearing animals. The results of this experiment demonstrate a novel preventative therapy for cancer cachexia and a novel use of Exendin-4. Further research is necessary to determine the mechanisms through which Exendin-4 exerts these potent effects.

Characterization of the Yoshida sarcoma: a model of cancer cachexia.

PURPOSE: Cancer cachexia contributes significantly to morbidity and mortality in individuals with cancer. Currently, the mechanisms contributing to the development of cachexia are largely unknown, leading to a paucity of treatment and prevention options. Animal models are necessary in determining causal mechanisms and in testing potential treatments. While the Yoshida sarcoma has been utilized for more than 50 years, the cachexia syndrome produced by this model has not been well characterized in the literature. METHODS: Tumor allografts were subcutaneously implanted in male Sprague Dawley rats (n = 16) and allowed to grow for 23 days. Control animals (n = 16) received a sham surgery. All rats were monitored daily for the presence of hallmark cachexia symptoms. RESULTS: The results demonstrate the presence of decreased body weight gain, as well as lower levels of body adiposity and skeletal muscle mass, in tumor-bearing animals, as compared to controls. CONCLUSIONS: While a large tumor burden was reached, the extent of cachexia was similar to that which is observed in many individuals with cancer cachexia. Future experiments utilizing this model are encouraged to identify mechanisms and effective treatment and prevention strategies.

The role of insulin resistance in the development of muscle wasting during cancer cachexia.

BACKGROUND: Cancer cachexia is a complex syndrome associated with multiple metabolic abnormalities. Insulin resistance is present in many cancer patients and may be one mechanism through which muscle wasting occurs. METHODS AND RESULTS: The present review examines evidence in support of a role for insulin resistance in the development of muscle wasting during cancer cachexia and identifies areas for future research. Patients suffering from cancer cachexia tend to exhibit insulin resistance and improvements in insulin resistance have the potential to improve cachexia symptoms. In addition, evidence suggests that insulin resistance may occur prior to the onset of cachexia symptoms. CONCLUSIONS: Further investigation of the role of insulin resistance in cancer cachexia is needed. The use of translational research in this area is strongly encouraged, and has important implications for clinical research and the treatment and prevention of cancer cachexia.

Repeated gastric distension alters food intake and neuroendocrine profiles in rats.

The consumption of a large food bolus leads to stomach distension. Gastric distension potently signals the termination of a meal by stimulating gastric mechanoreceptors and activating neuroendocrine circuitry. The ability to terminate a meal is altered in disorders such as bulimia nervosa (BN), binge-eating disorder (BED) and certain subtypes of obesity in which large quantities of food are frequently ingested. When a large meal is consumed, the stomach is rapidly stretched. We modeled this rapid distension of the stomach in order to determine if the neuroendocrine abnormalities present in these disorders, including increased gastric capacit3y, leptin dysregulation, and alterations in neuropeptide Y (NPY), and proopiomelanocortin (POMC) expression, were influenced by the rapid stretch aspect of repeatedly consuming a large meal. To test the effects of repeated gastric distension (RGD) on neuroendocrine factors involved in energy homeostasis, a permanent intra-gastric balloon was implanted in rats, and briefly inflated daily for 4 weeks. Though body weights and daily food intakes remained equivalent in RGD and control rats, a significant delay in the onset of feeding was present during the first and second, but not the third and fourth weeks of inflations. Despite equivalent body weights and daily caloric consumption, RGD animals had significantly decreased leptin levels (p<0.05), and tended to have increased fasting arcuate NPY levels (p=0.08), which were suppressed more than control animals following food intake (control and RGD decreases from baseline were 184.95% and 257.42%, respectively). NPY expression in the nucleus of the solitary tract followed a similar pattern. These data demonstrate that the act of regularly distending the stomach can have effects on the regulation of energy balance that are independent from those related to caloric consumption, and may be related to disorders such as BN, BED, and certain types of obesity in which meal termination is impaired.

Glucose tolerance in response to a high-fat diet is improved by a high-protein diet.

Consumption of a high-fat (HF) diet results in insulin resistance and glucose intolerance. Weight loss is often recommended to reverse these metabolic alterations and the use of a high-protein (HP), low-carbohydrate diet is encouraged. In lean rats, consumption of a HP diet improves glycemic control. However, it is unknown whether this diet has a similar effectiveness in rodents with impaired glucose tolerance. Rats were fed a HF or a chow (CH) diet for 6 weeks and then switched to a HP diet or a CH or pair-fed (PF) to the amount of kcals consumed per day by the HP group. Following the diet switch, body weight gain was attenuated as compared to HF rats, and similar between HP, CH, and PF rats. Despite similar weight progression, HP and PF rats had a significant decrease in body fat after 2 weeks, as compared to HF rats. In contrast, CH rats did not show this effect. Glucose tolerance was attenuated more quickly in HP rats than in CH or PF rats. These results indicate that a HP diet may be more effective than a balanced diet for improving glycemic control in overweight individuals.

Sensitivity to the anorectic effects of leptin is retained in rats maintained on a ketogenic diet despite increased adiposity.

BACKGROUND: Rats maintained on a ketogenic diet (KD; 80% fat, 15% protein, 5% carbohydrate) have increased adiposity and leptin as compared to chow-fed controls (CH; 16% fat, 19% protein, 65% carbohydrate), although body weights and daily caloric intakes do not differ. METHODS: Rats maintained on a KD or CH were assessed for responsivity to intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) leptin. Hypothalamic gene expression was evaluated to determine the effects of KD on proopiomelanocortin (POMC) mRNA expression and components of the leptin-signaling system. RESULTS: Caloric intake by KD rats was decreased at a lower dose of i.p. leptin (100 microg) than was required to reduce intake by CH rats (leptin, caloric intake was reduced in KD rats as compared to intake following i.p. saline; p < 0.05). In a separate experiment to evaluate responsivity to i.c.v. leptin, the minimal dose of leptin required to significantly reduce 24-hour caloric intake did not differ between the groups. In the arcuate nucleus, POMC mRNA was elevated after a lower dose of i.c.v. leptin in KD rats (5 microg) than was required to increase POMC mRNA expression in CH rats (15 microg) or reduce caloric intake in either group. Finally, evaluation of the level of phosphorylated STAT3 (pSTAT3) in the arcuate and SOCS3 mRNA in the hypothalamus revealed significantly more pSTAT3-positive cells and increased SOCS3 mRNA expression at baseline for KD rats, compared to CH, neither of which was further increased following i.p. leptin administration. CONCLUSION: These data demonstrate that despite increased adiposity, leptin and markers of leptin resistance, responsivity to the anorectic effects of exogenous leptin is retainable during maintenance on a KD.

Adolescent activity-based anorexia increases anxiety-like behavior in adulthood.

Activity-based anorexia is a paradigm that induces increased physical activity, reduced food intake, and heightened activity of the hypothalamic-pituitary-adrenal axis in adult rats. To investigate whether experience with activity-based anorexia produced enduring effects on brain and behavior, female adolescent rats experienced activity-based anorexia during adolescence and were tested in adulthood for anxiety-like behavior on an elevated plus maze and in an open field. Analysis of elevated plus maze and open field behavior in adulthood revealed that rats that experienced activity-based anorexia during adolescence, but not rats that were simply food restricted, displayed increased anxiety-like behavior in adulthood. Plasma corticosterone and expression levels of corticotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus and in the central nucleus of the amygdala were significantly elevated in adult rats that had undergone activity-based anorexia in adolescence in response to the open field exposure, as compared to control rats. These data demonstrate enduring effects of adolescent activity-based anorexia on anxiety-like behavior and neuroendocrine factors critical in stress responsivity in adulthood. Furthermore, we demonstrate that activity-based anorexia during adolescence serves as a model whereby prolonged anxiety is induced, allowing for evaluation of the behavioral and neural correlates of mediating anxiety-like behaviors in adulthood.

Insulin sensitivity and glucose tolerance are altered by maintenance on a ketogenic diet.

Low-carbohydrate, ketogenic diets (KD) are frequently implemented in efforts to reduce or maintain body weight, although the metabolic effects of long-term exposure to this type of diet remain controversial. This study assessed the responsivity to peripheral and central insulin, glucose tolerance, and meal-induced effects of consuming a KD in the rat. After 8 wk of consuming chow or KD, caloric intake after peripheral or central insulin and insulin and glucose levels after a glucose challenge were assessed. In a separate group of rats, glucose and insulin responses to either a low- or high-carbohydrate test meal were measured. Finally, rats maintained on KD were switched back to a chow diet, and insulin sensitivity and glucose tolerance were evaluated to determine whether the effects of KD were reversible. Maintenance on KD resulted in decreased sensitivity to peripheral insulin and impaired glucose tolerance. Furthermore, consumption of a high-carbohydrate meal in rats that habitually consumed KD induced significantly greater insulin and glucose levels for an extended period of time, as compared with chow-fed controls. Responsivity to central insulin was heightened in KD rats and associated with increased expression levels of insulin receptor mRNA. Finally, returning to a chow diet rapidly reversed the effects of KD on insulin sensitivity and glucose tolerance. These data suggest that maintenance on KD negatively affects glucose homeostasis, an effect that is rapidly reversed upon cessation of the diet.