Sex differences in a mouse model of diet-induced obesity: the role of the gut microbiome.

BACKGROUND: Recent decades have seen an exponential rise in global obesity prevalence, with rates nearly doubling in a span of 40 years. A comprehensive knowledge base regarding the systemic effects of obesity is required to create new preventative and therapeutic agents effective at combating the current obesity epidemic. Previous studies of diet-induced obesity utilizing mouse models have demonstrated a difference in bodyweight gain by sex. In such studies, female mice gained significantly less weight than male mice when given the same high fat (HF) diet, indicating a resistance to diet-induced obesity. Research has also shown sex differences in gut microbiome composition between males and females, indicated to be in part a result of sex hormones. Understanding metabolic differences between sexes could assist in the development of new measures for obesity prevention and treatment. This study aimed to characterize sex differences in weight gain, plasma lipid profiles, fecal microbiota composition, and fecal short chain fatty acid levels. We hypothesized a role for the gut microbiome in these sex differences that would be normalized following microbiome depletion. METHODS: A mouse model was used to study these effects. Mice were divided into treatment groups by sex, diet, and presence/absence of an antibiotic cocktail to deplete genera in the gut microbiome. We hypothesized that sex differences would be present both in bodyweight gain and systemic measures of obesity, including hormone and circulating free fatty acid levels. RESULTS: We determined statistically significant differences for sex and/or treatment for the outcome measures. We confirm previous findings in which male mice gained significantly more weight than female mice fed the same high fat diet. However, sex differences persisted following antibiotic administration for microbiome depletion. CONCLUSIONS: We conclude that sex differences in the gut microbiome may contribute to sex differences in obesity, but they do not explain all of the differences.

Sex differences in response to a high fat, high sucrose diet in both the gut microbiome and hypothalamic astrocytes and microglia.

Objectives: Obesity is a major epidemic in our population and has emerged as a primary health concern. Consumption of a high fat, high sugar (HFHS) diet can specifically lead to gut dysbiosis, increased inflammation, and neuroinflammation. Interestingly, sex differences in the response to a HFHS diet are emerging. In this study, we investigated the effects of a HFHS diet compared to a low fat, low sugar (LFLS) diet in 8 week old male and female C57Bl/6 mice.Methods: The diet was administered for 14 weeks; body weights and food consumption were evaluated weekly.Results: Male and female mice fed the HFHS diet gained significantly more weight than LFLS-fed mice. However, in agreement with previous studies, males gained significantly more weight on the HFHS diet compared to females fed the same diet. Importantly, we determined significant sex and diet-induced differences to gut microbiome composition using next generation Illumina sequencing. We also observed significantly less astrocyte densitometry and no significant change to microglial morphology in the hypothalamus of Female HFHS compared to Female LFLS. On the other hand, Male HFHS revealed no change to hypothalamic astrogliosis, but increased microgliosis compared to Male LFLS.Discussion: In this study, we determined sex and diet-induced differences in both the gut and the brain, however, future studies will need to be performed in order to test the direct role of the gut microbiome to weight gain and neuroinflammation in male and female mice.

Sex Differences in Demand for Highly Palatable Foods: Role of the Orexin System.

BACKGROUND: The prevalence of eating disorders, including binge eating disorder, is significantly higher in women. These findings are mirrored by preclinical studies, which indicate that female rats have a higher preference for palatable food and show greater binge-like eating compared with male rats. METHODS: Here, we describe a novel within-session behavioral-economic paradigm that allows for the simultaneous measurement of the intake at null cost (Q0) and normalized demand elasticity (α) of 3 types of palatable food (low fat, high fat, and chocolate sucrose pellets) via demand curve analysis. In light of evidence that the orexin (hypocretin) system is critically involved in reward and feeding behaviors, we also examined the role of orexin function in sex differences of economic demand for palatable foods. RESULTS: The novel within-session behavioral-economic approach revealed that female rats have higher intake (demand) than males for all palatable foods at low cost (normalized to body weight) but no difference in intake at higher prices, indicating sex-dependent differences in the hedonic, but not motivational, aspects of palatable food. Immediately following behavioral-economic testing, we observed more orexin-expressing neurons and Fos expression (measure of recent neural activation) in these neurons in female rats compared with male rats. Moreover, the orexin-1 receptor antagonist SB334867 reduced both low- and high-cost intake for palatable food in both male and female rats. CONCLUSIONS: These findings provide evidence of higher demand at low prices for palatable food in females and indicate that these behavioral differences may be associated with sexual dimorphism in orexin system function.

Activation of medial hypothalamic orexin neurons during a Go/No-Go task.

Orexin neurons (Orx; also referred to as hypocretin) are found exclusively in the hypothalamus, and release the neuropeptides orexin A and orexin B (also referred to as hypocretin 1 and 2) throughout the CNS. With its widespread targets, the orexin system is involved in a number of functions including, but not limited to stress, reward, wakefulness, and food seeking. Our laboratory has previously proposed that the dorsomedial hypothalamus (DMH) and perifornical (PFA) orexin neurons function in stress and arousal whereas those in lateral hypothalamus (LH) participate in reward processes (Harris and Aston-Jones, 2006). In the current study, we compared Fos activation in orexin neurons located in medial hypothalamus (DMH and PFA) to those in LH during a Go/No-Go task for a highly palatable food reward, a task that would likely activate regions for arousal/attention as well as reward. The Go/No-Go paradigm is a useful behavioral tool to measure behavioral inhibition, impulsivity, learning, and reaction time. Our results revealed increased activation of medial hypothalamic orexin neurons correlated with greater accuracy on the Go/No-Go task. No correlation was found between Go/No-Go accuracy and activation of lateral hypothalamic orexin neurons. This study supports a functional dichotomy of medial vs lateral orexin neurons, and indicates a role for medial orexin neurons in behavioral performance that requires response inhibition.

Microglia morphology and proinflammatory signaling in the nucleus accumbens during nicotine withdrawal.

Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.

Oxytocin Reduces Cocaine Cued Fos Activation in a Regionally Specific Manner.

Background: Oxytocin may be a possible treatment for multiple neuropsychiatric disorders, including cocaine addiction. Little is known about the site-specific effects of oxytocin on various drug addiction-related brain regions. Furthermore, sexually dimorphic effects of oxytocin on neural function in the addiction circuit have not been established. Here, we studied Fos expression following cocaine-cued reinstatement in both male and female rats. Methods: Male and female rats underwent self-administration, extinction, and reinstatement tests. On test days, rats were given oxytocin or vehicle, and lever pressing was measured in response to conditioned cocaine cues. Rats were perfused and Fos staining measured in the central amygdala, medial prefrontal cortex, nucleus accumbens core, and subthalamic nucleus. Fos/oxytocin double labeling occurred in the paraventricular nucleus of the hypothalamus. Results: Rats reinstated to cocaine cues relative to extinction responding and oxytocin reduced cocaine seeking. Oxytocin combined with contingent cue presentations increased Fos+ oxytocin cell bodies within the paraventricular nucleus of the hypothalamus relative to vehicle. Fos expression robustly increased in the central amygdala following oxytocin administration. Oxytocin reversed cue-induced Fos expression in the medial prefrontal cortex, nucleus accumbens core, and subthalamic nucleus. Central oxytocin infusion also attenuated reinstated cocaine seeking. Conclusions: Oxytocin decreased reinstated cocaine seeking, increased Fos activation in the paraventricular nucleus of the hypothalamus and central amygdala, but normalized cue-induced Fos activation in the medial prefrontal cortex, nucleus accumbens core, and subthalamic nucleus, thereby demonstrating regionally specific activation patterns. No sex differences were seen for the effects of oxytocin on cocaine seeking and Fos activation, indicating that oxytocin acts on similar central neural circuits critical to reinstated cocaine seeking in both males and females.

Detrimental effects of a high fat/high cholesterol diet on memory and hippocampal markers in aged rats.

High fat diets have detrimental effects on cognitive performance, and can increase oxidative stress and inflammation in the brain. The aging brain provides a vulnerable environment to which a high fat diet could cause more damage. We investigated the effects of a high fat/high cholesterol (HFHC) diet on cognitive performance, neuroinflammation markers, and phosphorylated Tau (p-Tau) pathological markers in the hippocampus of Young (4-month old) versus Aged (14-month old) male rats. Young and Aged male Fisher 344 rats were fed a HFHC diet or a normal control diet for 6 months. All animals underwent cognitive testing for 12days in a water radial arm maze to assess spatial and working reference memory. Hippocampal tissue was analyzed by immunohistochemistry for structural changes and inflammation, and Western blot analysis. Young and Aged rats fed the HFHC diet exhibited worse performance on a spatial working memory task. They also exhibited significant reduction of NeuN and calbindin-D28k immunoreactivity as well as an increased activation of microglial cells in the hippocampal formation. Western blot analysis of the hippocampus showed higher levels of p-Tau S202/T205 and T231 in Aged HFHC rats, suggesting abnormal phosphorylation of Tau protein following the HFHC diet exposure. This work demonstrates HFHC diet-induced cognitive impairment with aging and a link between high fat diet consumption and pathological markers of Alzheimer's disease.

Estrogen receptor alpha deficiency protects against development of cognitive impairment in murine lupus.

BACKGROUND: One of the more profound features of systemic lupus erythematosus (SLE) is that females have a 9:1 prevalence of this disease over males. Up to 80% of SLE patients have cognitive defects or affective disorders. The mechanism of CNS injury responsible for cognitive impairment is unknown. We previously showed that ERα deficiency significantly reduced renal disease and increased survival in lupus-prone mice. We hypothesized that ERα deficiency would be similarly protective in the brain, and that ERα may play a role in modulating blood-brain barrier (BBB) integrity and/or neuroinflammation in lupus-prone mice. METHODS: MRL/lpr ERα+/+ and ERαKO mice (n = 46) were ovariectomized, received 17ß-estradiol pellets, and underwent radial arm water maze (WRAM) and novel object recognition (NOR) testing starting at eight weeks of age. Mice were sacrificed and brains were hemisected and processed for either immunohistochemistry, or hippocampus and parietal cortex dissection for Western blotting. RESULTS: MRL/lpr ERαKO mice (n = 21) performed significantly better in WRAM testing than wild-type MRL/lpr mice (n = 25). There was a significant reduction in reference memory errors (P <0.007), working memory errors (P <0.05), and start arm errors (P <0.02) in ERαKO mice. There were significant differences in NOR testing, particularly total exploration time, with ERα deficiency normalizing behavior. No significant differences were seen in markers of tight junction, astrogliosis, or microgliosis in the hippocampus or cortex by Western blot, however, there was a significant reduction in numbers of Iba1+ activated microglia in the hippocampus of ERαKO mice, as evidenced by immunohistochemietry (IHC). CONCLUSION: ERα deficiency provides significant protection against cognitive deficits in MRL/lpr mice as early as eight weeks of age. Additionally, the significant reduction in Iba1+ activated microglia in the MRL/lpr ERαKO mice was consistent with reduced inflammation, and may represent a biological mechanism for the cognitive improvement observed.

Damaging effects of a high-fat diet to the brain and cognition: a review of proposed mechanisms.

The prevalence of obesity is growing and now includes at least one-third of the adult population in the United States. As obesity and dementia rates reach epidemic proportions, an even greater interest in the effects of nutrition on the brain have become evident. This review discusses various mechanisms by which a high fat diet and/or obesity can alter the brain and cognition. It is well known that a poor diet and obesity can lead to certain disorders such as type II diabetes, metabolic syndrome, and heart disease. However, long-term effects of obesity on the brain need to be further examined. The contribution of insulin resistance and oxidative stress is briefly reviewed from studies in the current literature. The role of inflammation and vascular alterations are described in more detail due to our laboratory's experience in evaluating these specific factors. It is very likely that each of these factors plays a role in diet-induced and/or obesity-induced cognitive decline.

Prolonged diet induced obesity has minimal effects towards brain pathology in mouse model of cerebral amyloid angiopathy: implications for studying obesity-brain interactions in mice.

Cerebral amyloid angiopathy (CAA) occurs in nearly every individual with Alzheimer's disease (AD) and Down's syndrome, and is the second largest cause of intracerebral hemorrhage. Mouse models of CAA have demonstrated evidence for increased gliosis contributing to CAA pathology. Nearly two thirds of Americans are overweight or obese, with little known about the effects of obesity on the brain, although increasingly the vasculature appears to be a principle target of obesity effects on the brain. In the current study we describe for the first time whether diet induced obesity (DIO) modulates glial reactivity, amyloid levels, and inflammatory signaling in a mouse model of CAA. In these studies we identify surprisingly that DIO does not significantly increase Aß levels, astrocyte (GFAP) or microglial (IBA-1) gliosis in the CAA mice. However, within the hippocampal gyri a localized increase in reactive microglia were increased in the CA1 and stratum oriens relative to CAA mice on a control diet. DIO was observed to selectively increase IL-6 in CAA mice, with IL-1ß and TNF-α not increased in CAA mice in response to DIO. Taken together, these data show that prolonged DIO has only modest effects towards Aß in a mouse model of CAA, but appears to elevate some localized microglial reactivity within the hippocampal gyri and selective markers of inflammatory signaling. These data are consistent with the majority of the existing literature in other models of Aß pathology, which surprisingly show a mixed profile of DIO effects towards pathological processes in mouse models of neurodegenerative disease. The importance for considering the potential impact of ceiling effects in pathology within mouse models of Aß pathogenesis, and the current experimental limitations for DIO in mice to fully replicate metabolic dysfunction present in human obesity, are discussed. This article is part of a Special Issue entitled: Animal Models of Disease.

NOX2 deficiency attenuates markers of adiposopathy and brain injury induced by high-fat diet.

The consumption of high-fat/calorie diets in modern societies is likely a major contributor to the obesity epidemic, which can increase the prevalence of cancer, cardiovascular disease, and neurological impairment. Obesity may precipitate decline via inflammatory and oxidative signaling, and one factor linking inflammation to oxidative stress is the proinflammatory, pro-oxidant enzyme NADPH oxidase. To reveal the role of NADPH oxidase in the metabolic and neurological consequences of obesity, the effects of high-fat diet were compared in wild-type C57Bl/6 (WT) mice and in mice deficient in the NAPDH oxidase subunit NOX2 (NOX2KO). While diet-induced weight gains in WT and NOX2KO mice were similar, NOX2KO mice had smaller visceral adipose deposits, attenuated visceral adipocyte hypertrophy, and diminished visceral adipose macrophage infiltration. Moreover, the detrimental effects of HFD on markers of adipocyte function and injury were attenuated in NOX2KO mice; NOX2KO mice had improved glucose regulation, and evaluation of NOX2 expression identified macrophages as the primary population of NOX2-positive cells in visceral adipose. Finally, brain injury was assessed using markers of cerebrovascular integrity, synaptic density, and reactive gliosis, and data show that high-fat diet disrupted marker expression in WT but not NOX2KO mice. Collectively, these data indicate that NOX2 is a significant contributor to the pathogenic effects of high-fat diet and reinforce a key role for visceral adipose inflammation in metabolic and neurological decline. Development of NOX-based therapies could accordingly preserve metabolic and neurological function in the context of metabolic syndrome.

Obesity increases cerebrocortical reactive oxygen species and impairs brain function.

Nearly two-thirds of the population in the United States is overweight or obese, and this unprecedented level of obesity will undoubtedly have a profound impact on overall health, although little is currently known about the effects of obesity on the brain. The objective of this study was to investigate cerebral oxidative stress and cognitive decline in the context of diet-induced obesity (DIO). We demonstrate for the first time that DIO induces higher levels of reactive oxygen species (ROS) in the brain and promotes cognitive impairment. Importantly, we also demonstrate for the first time in these studies that both body weight and adiposity are tightly correlated with the level of ROS. Interestingly, ROS were not correlated with cognitive decline in this model. Alterations in the antioxidant/detoxification Nrf2 pathway, superoxide dismutase, and catalase activity levels were not significantly altered in response to DIO. However, a significant impairment in glutathione peroxidase was observed in response to DIO. Taken together, these data demonstrate for the first time that DIO increases the levels of total and individual ROS in the brain and highlight a direct relationship between the amount of adiposity and the level of oxidative stress within the brain. These data have important implications for understanding the negative effects of obesity on the brain and are vital to understanding the role of oxidative stress in mediating the effects of obesity on the brain.

Role of physiological levels of 4-hydroxynonenal on adipocyte biology: implications for obesity and metabolic syndrome.

Lipid peroxidation products such as 4-hydroxynonenal (HNE) are known to be increased in response to oxidative stress, and are known to cause dysfunction and pathology in a variety of tissues during periods of oxidative stress. The aim of the current study was to determine the chronic (repeated HNE exposure) and acute effects of physiological concentrations of HNE toward multiple aspects of adipocyte biology using differentiated 3T3-L1 adipocytes. Our studies demonstrate that acute and repeated exposure of adipocytes to physiological concentrations of HNE is sufficient to promote subsequent oxidative stress, impaired adipogenesis, alter the expression of adipokines, and increase lipolytic gene expression and subsequent increase in free fatty acid (FFA) release. These results provide an insight in to the role of HNE-induced oxidative stress in regulation of adipocyte differentiation and adipose dysfunction. Taken together, these data indicate a potential role for HNE promoting diverse effects toward adipocyte homeostasis and adipocyte differentiation, which may be important to the pathogenesis observed in obesity and metabolic syndrome.

Mutant amyloid precursor protein differentially alters adipose biology under obesogenic and non-obesogenic conditions.

Mutations in amyloid precursor protein (APP) have been most intensely studied in brain tissue for their link to Alzheimer's disease (AD) pathology. However, APP is highly expressed in a variety of tissues including adipose tissue, where APP is also known to exhibit increased expression in response to obesity. In our current study, we analyzed the effects of mutant APP (E693Q, D694N, K670N/M671L) expression toward multiple aspects of adipose tissue homeostasis. These data reveal significant hypoleptinemia, decreased adiposity, and reduced adipocyte size in response to mutant APP, and this was fully reversed upon high fat diet administration. Additionally, mutant APP was observed to significantly exacerbate insulin resistance, triglyceride elevations, and macrophage infiltration of adipose tissue in response to a high fat diet. Taken together, these data have significant implications for linking mutant APP expression to adipose tissue dysfunction and global changes in endocrine and metabolic function under both obesogenic and non-obesogenic conditions.

Oxidative stress and cerebral endothelial cells: regulation of the blood-brain-barrier and antioxidant based interventions.

While numerous lines of evidence point to increased levels of oxidative stress playing a causal role in a number of neurodegenerative conditions, our current understanding of the specific role of oxidative stress in the genesis and/or propagation of neurodegenerative diseases remains poorly defined. Even more challenging to the "oxidative stress theory of neurodegeneration" is the fact that many antioxidant-based clinical trials and therapeutic interventions have been largely disappointing in their therapeutic benefit. Together, these factors have led researchers to begin to focus on understanding the contribution of highly localized structures, and defined anatomical features, within the brain as the sites responsible for oxidative stress-induced neurodegeneration. This review focuses on the potential for oxidative stress within the cerebrovascular architecture serving as a modulator of neurodegeneration in a variety of pathological settings. In particular, this review highlights important implications for vascular-derived oxidative stress in the initiating and promoting pathophysiology in the brain, identifying new roles for cerebrovascular oxidative stress in a variety of brain disorders. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Vascular changes in rat hippocampus following a high saturated fat and cholesterol diet.

The long-term effects of a diet rich in saturated fat and cholesterol on the hippocampus were evaluated in this study. It has previously been shown that this type of diet is detrimental to health, particularly affecting peripheral organs such as the heart and liver. However, effects on the brain have not been fully evaluated. This study focused on the hippocampus, a brain region instrumental for learning and memory and vulnerable to ischemic damage. Reduced blood-brain barrier (BBB) integrity and increased microgliosis were observed in the hippocampus of rats fed a high-saturated-fat and cholesterol (HFHC) diet for 6 months. Interestingly, an increase in hippocampal protein levels of occludin, a tight junction protein, was found in HFHC-treated rats as well. Further investigation revealed decreased expression of the occludin protein in blood vessels and increased expression in the dentate gyrus hilar neurons and mossy fibers of the hippocampal cornus ammonis 3 in HFHC-treated rats. Our results show alterations in BBB integrity and expression of tight junction proteins after long-term exposure to HFHC diet in rats. These findings may suggest a biologic mechanism for previously observed behavioral deficits occurring in rats fed this diet.

Diet-induced effects on neuronal and glial elements in the middle-aged rat hippocampus.

Consumption of a high-fat and/or high-cholesterol diet can have detrimental effects on the brain. In the present study, dietary treatment with saturated fats, trans fats, or cholesterol to middle-aged Fischer 344 rats resulted in alterations to serum triglyceride and cholesterol levels, organ weights, and hippocampal morphology. Previously, we demonstrated that a 10% hydrogenated coconut oil and 2% cholesterol diet resulted in worse performance on the 12-day water radial arm maze, increased cholesterol and triglyceride levels, and decreased dendritic microtubule associated protein 2 (MAP2) staining in the hippocampus. The diets administered herein were used to examine components from the previous diet and further examine their effects on hippocampal morphology. Specifically, neuronal morphology, dendritic integrity, fatty acid metabolism, microgliosis, and blood vessel structure in the hippocampus and/or adjacent structures were explored. Our results indicate alterations to peripheral and neural systems following each of the diets.

Effects of a saturated fat and high cholesterol diet on memory and hippocampal morphology in the middle-aged rat.

Diets rich in cholesterol and/or saturated fats have been shown to be detrimental to cognitive performance. Therefore, we fed a cholesterol (2%) and saturated fat (hydrogenated coconut oil, Sat Fat 10%) diet to 16-month old rats for 8 weeks to explore the effects on the working memory performance of middle-aged rats. Lipid profiles revealed elevated plasma triglycerides, total cholesterol, HDL, and LDL for the Sat-Fat group as compared to an iso-caloric control diet (12% soybean oil). Weight gain and food consumption were similar in both groups. Sat-Fat treated rats committed more working memory errors in the water radial arm maze, especially at higher memory loads. Cholesterol, amyloid-beta peptide of 40 (Abeta40) or 42 (Abeta42) residues, and nerve growth factor in cortical regions was unaffected, but hippocampal Map-2 staining was reduced in rats fed a Sat-Fat diet, indicating a loss of dendritic integrity. Map-2 reduction correlated with memory errors. Microglial activation, indicating inflammation and/or gliosis, was also observed in the hippocampus of Sat-Fat fed rats. These data suggest that saturated fat, hydrogenated fat and cholesterol can profoundly impair memory and hippocampal morphology.