Hypothalamic serotonin-insulin signaling cross-talk and alterations in a type 2 diabetic model.

Serotonin and insulin are key regulators of homeostatic mechanisms in the hypothalamus. However, in type 2 diabetes, the hypothalamic responsiveness to serotonin is not clearly established. We used a diabetic model, the Goto Kakizaki (GK) rats, to explore insulin receptor expression, insulin and serotonin efficiency in the hypothalamus and liver by means of Akt phosphorylation. Insulin or dexfenfluramine (stimulator of serotonin) treatment induced Akt phosphorylation in Wistar rats but not in GK rats that exhibit down-regulated insulin receptor. Studies in a neuroblastoma cell line showed that serotonin-induced Akt phosphorylation is PI3-kinase dependent. Finally, in response to food intake, hypothalamic serotonin release was reduced in GK rats, indicating impaired responsiveness of this neurotransmitter. In conclusion, hypothalamic serotonin as insulin efficiency is impaired in diabetic GK rats. The insulin-serotonin cross-talk and impairment observed is one potential key modification in the brain during the onset of diabetes.

Assessment of α-synuclein secretion in mouse and human brain parenchyma.

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.

Chronic central leptin infusion differently modulates brain and liver insulin signaling.

Recent studies reported the impact of leptin on peripheral insulin sensitivity and glucose utilization. However, little is known concerning the effect of central leptin on hypothalamic and hepatic insulin efficiency. This study aimed to determine the consequence of chronic intra-cerebroventricular (ICV) leptin or murine leptin antagonist (MLA) infusion on hypothalamic and hepatic insulin signaling pathways, in rats. A 2-week central leptin infusion enhanced insulin-dependent Akt phosphorylation in the liver without changing PTP-1B protein expression, associated to insulin receptor (IR) upregulation and reduced IRS-1 phosphorylation on Ser302 residue. In the hypothalamus, a chronic ICV leptin infusion induced PTP-1B associated with a specific decrease in insulin-dependent Akt phosphorylation. In contrast, a chronic MLA infusion did not alter IR and PTP-1B expressions in hypothalamus and liver. Our results underline a brain leptin-dependent increase in hepatic insulin efficiency as mirrored by IR up-regulation, increased insulin-dependent Akt phosphorylation and reduced IRS-1 phosphorylation on Ser302 residue.

High-fat feeding influences the endocrine responses of pubertal rats to an acute stress.

BACKGROUND/AIMS: Studies conducted in adult rats have shown that increased fat intake affects brain energy homeostasis and stress response. The neuroendocrine circuits controlling the aforementioned functions continue to mature during puberty. The aim of the present study was to investigate whether post-weaning high-fat consumption can modify the endocrine responses of pubertal rats to an acute stress. METHODS: Weaning male and female Wistar rats on postnatal day (P) 22 were fed either a high-fat (HF; 45% calories from fat) or a control (10% calories from fat) diet and were sacrificed on the individual day of puberty onset (between P35 and P42) under basal conditions or 4 h after swimming stress. Plasma insulin, leptin and corticosterone levels were determined by radioimmunoassay and the respective receptors in the hypothalamus and hippocampus were determined by Western blot analysis. RESULTS: Stressed HF-fed males showed a smaller increase in plasma insulin levels than chow-fed males. Their leptin receptor levels were reduced in the hypothalamus, but not in the hippocampus, and their glucocorticoid receptor levels were increased in the hypothalamus compared to stressed chow-fed males. HF-fed females were nonresponsive to stress-induced alterations in plasma glucose and corticosterone levels, as well as to hippocampal insulin receptors following stress. Several sex differences were also revealed in the endocrine responses of HF-fed animals following stress. CONCLUSIONS: These data show that consumption of high-fat foods during preadolescence can modify the endocrine responses to an acute stress by affecting both stress and metabolic mediators in a sexually dimorphic manner.

Fat feeding of rats during pubertal growth leads to neuroendocrine alterations in adulthood.

Juvenile obesity is a rising epidemic due largely to consumption of caloric dense, fat-enriched foods. Nevertheless, literature on fat-induced neuroendocrine and metabolic disturbances during adolescence, preceding obesity, is limited. This study aimed to examine early events induced by a fat diet (45% calories from saturated fat) in male rats fed the diet during the pre- and post-pubertal period. The neuroendocrine endpoints studied were the levels of circulating leptin, insulin and corticosterone, as well as their receptors in the hypothalamus and hippocampus. Hormonal levels were determined by radioimmunoassay and receptors' levels by western blot analysis. Leptinemia was increased in pubertal rats and in adult rats fed the fat diet from weaning to adulthood, but not in those fed from puberty to adulthood. Modifications in the developmental pattern from puberty to adulthood were observed for most of the brain receptors studied. In adult animals fed the fat diet from weaning onwards, the levels of leptin receptors in the hypothalamus and glucocorticoid receptors in the hippocampus were decreased compared to chow-fed controls. Switching from fat to normal chow at puberty onset restored the diet-induced alterations on circulating leptin, but not on its hypothalamic receptors. These data suggest that when a fat-enriched diet, resembling those consumed by many teenagers, provided in rats during pubertal growth, it can longitudinally influence the actions of leptin and corticosterone in the brain. The observed alterations at a preobese state may constitute early signs of the disturbed energy balance toward overweight and obesity.

Adult consequences of post-weaning high fat feeding on the limbic-HPA axis of female rats.

The peripubertal period is critical for the final maturation of circuits controlling energy homeostasis and stress response. However, the consequence of juvenile fat consumption on adult physiology is not clear. This study analyzed the adult consequences of post-weaning fat feeding on limbic-hypothalamic-pituitary-adrenal (HPA) axis components and on metabolic regulators of female rats. Wistar rats were fed either a high fat (HF) diet or the normal chow from weaning to puberty or to 3 months of age. Additional groups crossed their diets at puberty onset. Plasma leptin, insulin, and corticosterone levels were determined by radioimmunoassay and their brain receptors by western blot analysis. Adult HF-fed animals though not overweight, had higher corticosterone and reduced glucocorticoid receptor levels in the hypothalamus and hippocampus, compared to the controls. The alterations in HPA axis emerged already at puberty onset. Leptin receptor levels in the hypothalamus were reduced only by continuous fat feeding from weaning to adulthood. The pre-pubertal period appeared more vulnerable to diet-induced alterations in adulthood than the post-pubertal one. Switching from fat diet to normal chow at puberty onset restored most of the diet-induced alterations in the HPA axis. The corticosteroid circuit rather than the leptin or insulin system appears as the principal target for the peripubertal fat diet-induced effects in adult female rats.

A dietary fat excess alters metabolic and neuroendocrine responses before the onset of metabolic diseases.

Early changes in neuroendocrine pathways are essential in the development of metabolic pathologies. Thus, it is important to have a better understanding of the signals involved in their initiation. Long-term consumption of high-fat diets induces insulin resistance, obesity, diabetes. Here, we have investigated early neural and endocrine events in the hypothalamus and hippocampus induced by a short-term high fat, low carbohydrate diet in adult male Wistar rats. The release of serotonin, which is closely associated with the actions of insulin and leptin, was measured, by electrochemical detection following reverse-phase liquid chromatography (HPLC), in the extracellular space of the medial hypothalamus and the dorsal hippocampus in samples obtained from non-anesthetized animals, by microdialysis. The high-fat diet had a specific effect on the hypothalamus. Serotonin release induced by food intake was reduced after 1 week, and effectively ceased after 6 weeks of the diet. After 1 week, there was an increased gene expression of the insulin receptor and the insulin receptor substrates IRS1 and IRS2, as measured by real-time PCR. After 6 weeks of diet, insulin gene expression increased. Leptinemia increased in all cases. This new data support the concept that high-fat diets, in addition to have peripheral effects, cause a rapid alteration in specific central mechanisms involved in energy and glucose homeostasis. The changes in the gene expression of insulin and signaling elements represent possible adaptations aimed at counterbalancing the reduced responsiveness of the serotonergic system to nutritional signals and maintaining homeostasis.

Fat diet affects leptin receptor levels in the rat cerebellum.

OBJECTIVE: The role of leptin receptors (Ob-Rs) within the hypothalamus in the control of energy expenditure has well been established. However, their role and regulation in other brain areas, including the cerebellum, is largely unexplored. In the present study we examined whether Ob-R levels in the rat cerebellum are influenced by a high-fat diet and if these changes are sexually divergent during adolescence. METHODS: The fat diet (45% energy from fat) was applied from weaning to puberty (postnatal days [P] 22-42), from weaning to adulthood (P22-90), and from puberty to adulthood (P42-90) in female and male Wistar rats. Ob-R levels were detected by western blotting and the data from pubertal and adult rats were analyzed by two-way analysis of variance for the effects of diet and sex. RESULTS: The fat diet affected Ob-R long isoform levels in a sexually dimorphic manner. In the cerebellum of all fat-fed male groups, Ob-R levels were reduced compared with their chow-fed counterparts (P < 0.05). In contrast, in female rat Ob-R levels were reduced only in the adult P22-90 group. CONCLUSION: Our data show for the first time that Ob-R levels in the rat cerebellum are subject to diet-induced alterations and that these changes are sexually dimorphic.

Brain insulin, energy and glucose homeostasis; genes, environment and metabolic pathologies.

The central nervous system is essential in maintaining energy and glucose homeostasis. In both animals and humans, efficient cerebral insulin signalling is a pivotal control element in these pathophysiological processes. The action of insulin in the brain is under a multilevel control via metabolic, endocrine and neural signals induced by nutrients, integrated mainly by the hypothalamus. Of particular interest is the interaction of insulin with the anabolic and catabolic neuroregulators. The anorexic peptides insulin, leptin and the neurotransmitter serotonin share common signalling pathways involved in food intake, in particular the insulin receptor substrate, phosphatidylinositol-3-kinase (PI3K) pathway. The dialogue of neurotransmitters and peptides via this signalling pathway is potentially of major importance in the pathophysiology of the brain in general and specifically in the regulation of feeding behaviour. At this time, a new concept in the aetiopathology of type 2 diabetes is immerging. This concept proposes that the combination of defective pancreatic beta-cell function and insulin resistance not only in classical insulin target tissues but in every tissue, contributes to the onset of the disease. It highlights the importance of the disruption of cerebral insulin signal transmission and its direct relation to metabolic diseases. Impaired brain insulin signalling, a link coupling obesity to diabetes, may be related to either genetic factors, or environmental factors such as stress, over or under-feeding and unbalanced diets: such factors may work either independently or in concert. Current approaches used for the prevention and treatment of type 2 diabetes are not adequately effective. Most of the anti-diabetic therapies induce many adverse effects, in particular obesity, and thus may initiate a vicious cycle of problems. In order to develop new, more efficient, preventive and therapeutic strategies for metabolic pathologies, there is an urgent need for increased understanding of the complexity of insulin signalling in the brain and on the interactive, central and peripheral effects of insulin.

Early neuroendocrine alterations in female rats following a diet moderately enriched in fat.

1. High-fat diets disrupt metabolic equilibrium and hypothalamic-pituitary-adrenal axis function and may lead to the development of metabolic and endocrine dysfunctions. The early neuroendocrine responses elicited by a combination of short-term metabolic and emotional stressors is not fully elucidated. 2. The purpose of the present study was to determine the impact on female rats, of a short-term enriched in fat diet, combined with an acute stressor. 3. Adult female Wistar rats were fed a fat diet for 7 days and subsequently exposed to 5 min swimming stress. Plasma leptin, insulin, glucose, luteinizing hormone (LH) and corticosterone, along with brain corticosteroid receptors' mRNAs were measured at 1 h post stress. 4. Diet, compared to chow, reduced food intake and body weight gain, increased leptin and LH, and decreased glucose in the periphery. The diet increased plasma corticosterone and reduced GR mRNA in the hippocampus, similarly to swim stress. 5. The diet significantly modified the animals' response to the subsequent swim stress, by blocking further corticosterone rise and GR mRNA reduction. In addition, exposure of diet-fed rats to stress, altered their endocrine response, in terms of leptin and LH. 6. These observations suggest that even short, moderately unbalanced diets can affect peripheral and central components of energy balance, reproduction and stress response.

Persisting neural and endocrine modifications induced by a single fat meal.

1. High-fat diets, modify the neuroendocrine response and, when prolonged, result in positive energy balance and obesity. Little is known about the effects of fat on the mechanisms operating in the initial steps of the neural and endocrine disturbances. 2. The studies reported here were designed to access the impact of the consumption of a single exclusively animal fat meal (lard), 24 h following its ingestion a) on the response of the hypothalamic serotonergic system to a standard laboratory chow meal and b) on the circulating levels of glucose, insulin, and leptin. The release of serotonin in the extracellular medial hypothalamic space (including the paraventricular-PVN and ventromedian-VMH nuclei) was determined using electrochemical detection following HPLC in samples obtained in vivo by microdialysis, in nonanesthetized adult male Wistar rats. 3. A lard meal resulted in decreased hypothalamic serotonin release postprandially and attenuated (24 h later) the hypothalamic serotonin response that normally follows a balanced meal. 4. In permanently catheterized rats, postprandial glucose and insulin levels measured in samples obtained in vivo, were either not, or only slightly, modified after a lard meal, whereas plasma leptin levels were increased. Interestingly, 24 h after a meal, insulin and leptin levels were increased in those animals eating a fat meal compared with those eating chow. Next-day glucose levels remained identical after the absorption either of a chow, or a lard meal. 5. The changes induced by the fat meal on peripheral and central regulators of energy and glucose homeostasis represent either adaptive mechanisms or early alterations that could render the organism vulnerable to further insults.

Neonatal handling and gender modulate brain monoamines and plasma corticosterone levels following repeated stressors in adulthood.

Neonatal handling affects the response to repeated stress in a sexually dimorphic manner. In order to elucidate the mechanisms underlying these gender-dependent effects, we investigated the consequences of neonatal androgenization and handling on adult stress reactivity by determining: (a) immobility time during repeated forced swimming, (b) plasma corticosterone levels, and (c) brain serotonin and dopamine levels and turnover after either repeated forced swimming, or repeated forced swimming followed by repeated restraint stress. In neonatally androgenized females, immobility time was lower in the handled than in the non-handled rats, a pattern resembling that of the males, suggesting that the sexually dimorphic effect of handling on immobility time can be attributed to the organizational effects of testosterone. No differences were found between androgenized females and females injected neonatally with vehicle, indicating that the gender differences in circulating corticosterone are not due to the organizational effects of testosterone. The stress of a neonatal injection interacted with neonatal handling resulting in lower plasma corticosterone and hypothalamic dopamine and serotonin levels in the neonatally injected handled animals following repeated forced swimming. The serotonergic system appears to be sensitive to both the organizational actions of testosterone and the effects of handling, since handled androgenized females had higher serotonin levels and decreased turnover following repeated forced swimming stress, compared to those injected neonatally with vehicle. Handling resulted in increased hypothalamic and striatal serotonin levels in both males and females following repeated forced swimming. Our results reveal that handling has gender-dependent effects on adult hypothalamic-pituitary-adrenal axis and brain monoaminergic system reactivity to stress and that these effects can be attributed to both the organizational and activational effects of gonadal hormones.

Impaired neuroendocrine response to stress following a short-term fat-enriched diet.

Unbalanced diets and stressful situations disrupt energy homeostasis and are implicated in the development of severe pathologies. The present study investigated the effects of a 7-day diet, enriched in corn oil (20%) and proportionally lower in protein and carbohydrate, on the major regulators of energy expenditure and stress response of adult male Wistar rats exposed to acute swimming stress at the end of the dietary treatment. Food intake and body weight gain were lower in diet-fed as compared with normal-chow-fed controls. The circulating leptin levels were elevated in both nonstressed and stressed diet-fed rats, while the glucose levels were significantly increased only in the diet-fed group subjected to stress. The plasma insulin levels were not affected by the diet, but were significantly reduced in acutely stressed rats. Acute swimming increased corticosterone levels both in chow-fed and diet-fed rats. No significant effect of diet was observed on corticosterone levels. Northern blot analysis showed increased glucocorticoid receptor mRNA levels in the hypothalamus of normally fed rats subjected to stress. This increase was not observed in the diet-fed stressed group, which on the contrary showed reduced glucocorticoid receptor mRNA levels following stress. The data presented indicate that even a moderately unbalanced, fat-enriched diet can within a short time disrupt the metabolic neuroendocrine balance and the stress response, rendering the organism more vulnerable to potential stressful insults.

Effect of neonatal handling and sex on basal and chronic stress-induced corticosterone and leptin secretion.

Neonatal handling is an experimental paradigm for early experiences. It affects the programming of hypothalamo-pituitary-adrenal (HPA) axis function, known to be sexually dimorphic. Recently leptin, a hormone related to energy balance and secreted mainly by adipocytes, has been implicated in the stress response. We thus determined the effect of neonatal handling on plasma concentrations of corticosterone and leptin of male and female rats under basal conditions and after two consecutive chronic stressors: chronic forced swimming stress and chronic restraint. Handling resulted in lower basal corticosterone levels in both males and females and in a more efficient HPA response, with a large corticosterone surge following the first chronic stressor and a return to basal levels following the second. Handling also resulted in decreased plasma leptin concentrations in males, thus abolishing the sex difference in leptin levels. Furthermore, handling increased body weight while it decreased food intake under basal conditions. Food intake and body weight gain during chronic forced swimming was lower in handled than in non-handled males, while in females these parameters were not influenced by handling. In both males and females, handling resulted in decreased food intake and increased body weight loss during chronic restraint stress. Body weight loss during chronic restraint stress, which is considered an index of maladaptation and 'depression', was particularly pronounced in the handled females. Our results also showed that non-handled females had higher corticosterone and lower leptin levels than males under basal conditions and following each of the two chronic stressors. The present work suggests that early experiences, such as the mother-infant relationship, interact with endogenous factors, such as gonadal hormones, to determine the organism's response to stressful stimuli during adulthood.

Brain insulin: regulation, mechanisms of action and functions.

1. While many questions remained unanswered, it is now well documented that, contrary to earlier views, insulin is an important neuromodulator, contributing to neurobiological processes, in particular energy homeostasis and cognition. A specific role on cognitive functions related to feeding is proposed, and it is suggested that brain insulin from different sources might be involved in the above vital functions in health and disease. 2. A molecule identical to pancreatic insulin, and specific insulin receptors, are found widely distributed in the central nervous system networks related to feeding, reproduction, or cognition. 3. The actions of insulin in the central nervous system may be under both multilevel and multifactorial controls. The amount of blood insulin reaching the brain, brain insulin stores and secretion, potential local biosynthesis and degradation of the peptide, and insulin receptors and signal transduction can be affected by metabolic factors induced by nutrients, hormones, neurotransmitters, and regulatory peptides, peripherally or in the central nervous system. 4. Glucose and serotonin regulate insulin directly in the hypothalamus and may be of importance for its biological effects. Central mechanisms regulating glucose-induced insulin secretion show some analogy with the mechanisms operating in the pancreas. 5. A cross-talk between insulin and leptin receptors has been observed in the brain, and a regulation of central insulin actions, potentially via serotonin modulation, by leptin, galanin, melancortins, and neuropeptide Y (NPY) is suggested. 6. A more complete knowledge of the biological role of insulin in brain function and dysfunction, and of the regulatory mechanisms involved in these processes, constitutes a real advancement in the understanding of the pathophysiology of metabolic and mental diseases and could lead to important medical benefits.