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Based on studies delivering ghrelin or ghrelin receptor agonists, we have learned a great deal about the importance of the brain ghrelin signalling system for a wide range of physiological processes that include feeding behaviours, growth hormone secretion and glucose homeostasis. Because these processes can be considered as essential to life, the question arises as to why mouse models of depleted ghrelin signalling are not all skinny dwarfs with a host of behavioural and metabolic problems. Here, we provide a systematic detailed review of the phenotype of mice with deficient ghrelin signalling to help better understand the relevance and importance of the brain ghrelin signalling system, with a particular emphasis on those questions that remain unanswered.

The gravitostat is a novel homeostatic body weight-regulating mechanism, mostly studied in mice, and recently confirmed in obese humans. In the present study, we explored the effect of weight loading on metabolic outcomes, meal patterns and parameters linked to energy expenditure in both obese and lean rats. Diet-induced obese (DIO) and lean rats were implanted with capsules weighing either 15% of biological body weight (load) or empty capsules (1.3% of body weight; controls). Loading protected against fat accumulation more markedly in the DIO group. In line with this, the obesity-related impairment in insulin sensitivity was notably ameliorated in DIO rats upon loading, as revealed by the reduction in serum insulin levels and homeostatic model assessment for insulin resistance index scores. Although 24-hour caloric intake was reduced in both groups, this effect was greater in loaded DIO rats than in loaded lean peers. During days 10-16, after recovery from surgery, loading: (i) decreased meal size in both groups (only during the light phase in DIO rats) but this was compensated in lean rats by an increase in meal frequency; (ii) reduced dark phase locomotor activity only in lean rats; and (iii) reduced mean caloric efficiency in DIO rats. Muscle weight was unaffected by loading in either group. Dietary-obese rats are therefore more responsive than lean rats to loading.

The lateral parabrachial nucleus (lPBN), located in the pons, is a well-recognized anorexigenic center harboring, amongst others, the calcitonin gene-related peptide (CGRP)-expressing neurons that play a key role. The receptor for the orexigenic hormone ghrelin (the growth hormone secretagogue receptor, GHSR) is also abundantly expressed in the lPBN and ghrelin delivery to this site has recently been shown to increase food intake and alter food choice. Here we sought to explore whether GHSR-expressing cells in the lPBN (GHSRlPBN cells) contribute to feeding control, food choice and body weight gain in mice offered an obesogenic diet, involving studies in which GHSRlPBN cells were silenced. We also explored the neurochemical identity of GHSRlPBN cells. To silence GHSRlPBN cells, Ghsr-IRES-Cre male mice were bilaterally injected intra-lPBN with a Cre-dependent viral vector expressing tetanus toxin-light chain. Unlike control wild-type littermates that significantly increased in body weight on the obesogenic diet (i.e., high-fat high-sugar free choice diet comprising chow, lard and 9% sucrose solution), the heterozygous mice with silenced GHSRlPBN cells were resistant to diet-induced weight gain with significantly lower food intake and fat weight. The lean phenotype appeared to result from a decreased food intake compared to controls and caloric efficiency was unaltered. Additionally, silencing the GHSRlPBN cells altered food choice, significantly reducing palatable food consumption. RNAscope and immunohistochemical studies of the lPBN revealed considerable co-expression of GHSR with glutamate and pituitary adenylate cyclase-activating peptide (PACAP), and much less with neurotensin, substance P and CGRP. Thus, the GHSRlPBN cells are important for diet-induced weight gain and adiposity, as well as in the regulation of food intake and food choice. Most GHSRlPBN cells were found to be glutamatergic and the majority (76%) do not belong to the well-characterized anorexigenic CGRP cell population.

Chlorpyrifos (CPF) is one of the most commonly used organophosphate pesticides in the world. Our previous results described that apolipoprotein E (APOE) polymorphisms are a source of individual differences in susceptibility to CPF. The aim of this study was to assess the physical and biochemical effects of postnatal exposure to CPF in the apoE targeted replacement mouse model. Mice were exposed to CPF at 0 or 1 mg/kg/day from postnatal day 10–15. Physical development, plasma and forebrain cholinesterase (ChE) activity and gene expression in liver and forebrain were evaluated. CPF exposure delays physical maturation and decreases the expression of choline acetyltransferase, α4-subunit and the α7 receptor. CPF decreases the expression of vesicular acetylcholine transporter (VAChT) mRNA in the forebrain only in apoE3 mice. The expression of paraoxonase-2 in the forebrain was also influenced by APOE genotype and CPF. Differences between genotypes were observed in litter size, ChE activity, expression of butyrylcholinesterase and paraoxonase-1 in liver and variants of acetylcholinesterase, VAChT and the α7 receptor in the forebrain. These results support that there are different vulnerabilities to postnatal CPF exposure according to the APOE polymorphism, which in turn affects the cholinergic system and defenses to oxidative stress.

Recently, we have provided evidence, suggesting that mice expressing the human apolipoprotein E3 (apoE3) are more prone to develop an obesity-like phenotype and a diabetic profile when subchronically fed a chlorpyrifos (CPF)-supplemented diet. The aim of the current study was to examine the underlying mechanisms through which CPF alters both insulin- and leptin-signalling pathways in an APOE-dependent manner. Both adult apoE3- and E4-targeted replacement and C57BL/6 mice were exposed to CPF at 0 or 2 mg/kg body weight/day through the diet for 8 consecutive weeks. We determined the expression of JAK2, p-JAK2, STAT3, p-STAT3, SOCS3, IRS-1, p-IRS-1, AKT, p-AKT, GSK3β, p-GSK3β, and apoE in the liver, as well as hepatic mRNA levels of pon1, pon2, and pon3. CPF markedly disrupted both leptin and insulin homeostasis, particularly in apoE3 mice. Indeed, only CPF-fed apoE3 mice exhibited an increased phosphorylation ratio of STAT3, as well as increased total SOCS3 protein levels. Similarly, the exposure to CPF drastically reduced the phosphorylation ratio of both AKT and GSK3β, especially in apoE3 mice. Overall, CPF reduced the expression of the three pon genes, principally in C57BL/6 and apoE3 mice. These results provide notable mechanistic insights on the metabolic effects of the pesticide CPF, and attest the increased vulnerability of apoE3 carriers to its metabolic-disruptor role.

To date, we have shown that apolipoprotein E (APOE) polymorphisms differentially modulate the neurobehavioral and metabolic effects of chlorpyrifos (CPF), a widely used pesticide, which is detected as residue in food. We previously reported that, after being exposed to CPF, APOE3 subjects exhibit metabolic dysfunctions while APOE4 subjects undergo changes in behavior. In the current study, we investigated the effects of a double exposure to CPF on social behavior and hypothalamic gene expression in apoE-targeted replacement (TR) mice. Male apoE3-and apoE4-TR mice were exposed to CPF at 0 or 1 mg/kg/day on postnatal days 10–15 and then, during adulthood (5 months of age), fed a CPF-supplemented diet (0 or 2 mg/kg/day) for 15 days. During adult exposure to CPF, body weight gain and food intake were monitored. At the end of the adult exposure period, we evaluated social behavior in a three-chamber test, as well as mRNA levels of hypothalamic neuropeptides and receptors related to social behavior and feeding control. Adult CPF exposure increased food intake in general, but only apoE4 mice increased their body weight. Postnatal CPF exposure improved preference for the social contexts in apoE4 mice while adult CPF exposure did the same in apoE3 mice. Anorexigenic-peptide and social-related behavior gene expression decreased as a result of adult CPF exposure in apoE4 mice, and neuropeptide Y was more expressed in apoE4 mice. These results indicate that CPF exposure produces orexigenic and metabolic effects and enlarges individual differences in social behavior, especially in apoE3 mice.

Developmental exposure to toxicants and diet can interact with an individual&rsquo, s genetics and produce long-lasting metabolic adaptations. The different isoforms of the apolipoprotein E (APOE) are an important source of variability in metabolic disorders and influence the response to the pesticide chlorpyrifos (CPF). We aimed to study the epigenetic regulation on feeding control genes and the influence of postnatal CPF exposure, APOE genotype, and sex, and how these modifications impact on the metabolic response to a high-fat diet (HFD). Both male and female apoE3- and apoE4-TR mice were exposed to CPF on postnatal days 10&ndash, 15. The DNA methylation pattern of proopiomelanocortin, neuropeptide Y, leptin receptor, and insulin-like growth factor 2 was studied in the hypothalamus. At adulthood, the mice were given a HFD for eight weeks. The results highlight the importance of sex in the epigenetic regulation and the implication of CPF treatment and APOE genotype. The body weight progression exhibited sex-dimorphic differences, apoE4-TR males being the most susceptible to the effects induced by CPF and HFD. Overall, these results underscore the pivotal role of sex, APOE genotype, and developmental exposure to CPF on subsequent metabolic disturbances later in life and show that sex is a key variable in epigenetic regulation.

The apolipoprotein E (APOE) e4 allele hastens cognitive decline, but other non-cognitive behaviours, as well as underpinning interactions with the cholinergic system, have not been systematically addressed. Both C57BL/6 and humanised apoE4 female mice were transiently exposed to subclinical doses (0 or 1 mg/kg body weight) of the cholinesterase inhibitor chlorpyrifos (CPF), a widely-used pesticide, from postnatal days 10–15. At 5 months of age, we assessed the impact of APOE4 genotype, postnatal CPF exposure and APOE4 x CPF interactions on anxiety (open field and light-dark tests), stereotypes (digging test) and neophobia (sucrose preference test), as well as on high-fat diet (HFD)-seeking and consumption (scheduled-feeding paradigm). We found that control APOE4 female carriers displayed a robust anxiety-like phenotype, which was accompanied by exaggerated stereotypes and a subtle neophobic response to rewarding foods. In parallel, we observed an amplified “wanting” response for HFD in these mice, which did not entail enhanced “liking”. Notably, postnatal CPF ameliorated the anxiety-like and the heightened HFD-seeking responses in adult apoE4 female mice, while caused them to gain weight steadily compared to control peers. In turn, an early-life transient exposure to CPF fostered the over-consumption of HFD during adulthood without affecting how much this reward was “wanted” or the total caloric intake. These data reveal a role for CPF towards fostering “unhealthy” dietary choices. We conclude that the APOE4 genotype modulates non-cognitive behaviours and we provide support for an APOE4-dependent cholinergic dysfunction.

Polymorphisms of the apolipoprotein E (APOE) gene differentially affect neurobiological functions and cognitive performance and confer different vulnerabilities to subclinical exposures to chlorpyrifos (CPF), a pesticide used worldwide. The data reported on this topic suggest a complex interaction between cholinergic signaling and the APOE genotype. To gain greater functional insight into this interaction, we evaluated spatial learning and memory and hippocampal cholinergic expression in young apoE3 and apoE4 transgenic mice exposed to CPF. Male and female mice were exposed to CPF at 0 or 1 mg/kg on postnatal days 10–15 and then, exposed to CPF at 0 or 2 mg/kg for 60 days at 5 months of age. At 6 months of age, mice were tested for spatial skills in a Barnes maze. At the end of the task, animals were killed and gene expression of cholinergic components was assessed in the hippocampus. Our results show that apoE4 female mice performed worse in the spatial task, while postnatal CPF impaired escape strategies and spatial memory in apoE3 mice. In turn, CPF in adulthood improved spatial abilities in apoE4 female mice. Regarding gene expression, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) expression were increased in apoE4 mice. Postnatal exposure to CPF increased ChAT mRNA levels in apoE4 mice, whereas adult exposure to CPF induced changes in acetylcholinesterase-S, α7- and α4-subunit nicotinic receptor expression in apoE4 females. The current findings provide new insights into APOE-dependent cholinergic signaling, which directly affects the response to CPF cholinergic insult, especially in APOE4 subjects.

Increasing evidence links the widespread exposure to organophosphate (OP) pesticides to the global epidemics of type 2 diabetes and obesity. Our recent data highlighted gene×environment interactions: mice expressing the human apolipoprotein E3 (apoE3) isoform were more prone to develop obesity than those expressing apoE2 or apoE4 upon dietary challenge with chlorpyrifos (CPF), the most used OP worldwide. Thus, we aimed to further explore the contribution of the APOE3 genotype on the emergence of obesity and related metabolic dysfunctions upon subchronic exposure to CPF. Seven-month-old targeted replacement apoE3 and C57BL/6N male mice were orally exposed to CPF at 0 or 2mg/kg body weight/day for 8 consecutive weeks. We examined body weight status, food and water intake, lipid and glucose homeostasis, metabolic biomarkers concentrations, insulin levels and insulin resistance, and leptin and ghrelin profiles. CPF exposure generally increased food ingestion, glucose and total cholesterol concentrations, and tended to elevate acyl ghrelin levels. Nonetheless, excess weight gain and increased leptin levels were inherent to apoE3 mice. Moreover, the propensity towards a diabetic profile was markedly higher in these animals than in C57BL/6N, as they showed a higher homeostatic model assessment for insulin resistance index and higher insulin levels. Although both genotypes were metabolically affected by CPF, the results of the present investigation revealed that apoE3 mice were the most vulnerable to developing obesity and related disturbances following CPF administration through the diet. Since the APOE3 genotype is the most prevalent worldwide, current findings have particular implications for human health.

Rationale: The apolipoprotein E (apoE) genotype influences cognitive performance in humans depending on age and sex. While the detrimental role of the apoE4 isoform on spatial learning and memory has been well-established in humans and rodents, less is known on its impact on the executive functions. Objectives: We aimed to evaluate the effect of apoE isoforms (apoE2, apoE3, apoE4) on visuospatial attention and inhibitory control performance in female transgenic mice, and to determine the neurochemical and neuropharmacological basis of this potential relationship. Methods: Female mice carrying apoE2, apoE3, and apoE4 were trained in the five-choice serial reaction time task (5-CSRTT). Upon a stable performance, we manipulated the inter-trial interval and the stimulus duration to elicit impulsive responding and engage attention respectively. We further performed a pharmacological challenge by administering cholinergic and GABAergic agents. Finally, we analyzed the levels of brain amino acids and monoamines by using reversed phase high-performance liquid chromatography (HPLC). Results: ApoE4 mice showed a deficient inhibitory control as revealed by increased perseveration and premature responding. When attention was challenged, apoE4 mice also showed a higher drop in accuracy. The adverse effect of scopolamine on the task was attenuated in apoE4 mice compared to apoE2 and apoE3. Furthermore, apoE4 mice showed less dopamine in the frontal cortex than apoE2 mice. Conclusions: We confirmed that the apoE genotype influences attention and inhibitory control in female transgenic mice. The influence of apoE isoforms in the brain neuromodulatory system may explain the cognitive and behavioral differences attributable to the genotype., This research was supported by PSI2010-21743-C02-01, the Ministry of the Economy and Competitiveness (MINECO, Spain), and PI 13/01252 from the Instituto de Salud Carlos III (ISCIII, Spain), the European Regional Development Fund (ERDF), the Commission for Universities and Research of the Department of Innovation, Universities and Enterprise of the Generalitat de Catalunya (2009 FI-B 00256/2010 and BE-1 0072), and the European Social Fund