A free-choice high-fat diet modulates the effects of a sucrose bolus on the expression of genes involved in glucose handling in the hypothalamus and nucleus accumbens.

The consumption of saturated fat and sucrose can have synergistic effects on the brain that do not occur when either nutrient is consumed by itself. In this study we hypothesize that saturated fat intake modulates glucose handling in the hypothalamus and nucleus accumbens, both brain areas highly involved in the control of food intake. To study this, male Wistar rats were given a free-choice high fat diet (fcHFD) or a control diet for two weeks. During the last seven days rats were given a daily bolus of either a 30% sucrose solution or water. Rats were sacrificed on day eight, 30 minutes after the onset of drinking. mRNA and protein levels of genes involved in glucose handling were assessed in the hypothalamus and nucleus accumbens. We found increased Glut3 and Glut4 mRNA in the hypothalamus of fcHFD-fed rats without an additional effect of the sucrose bolus. In the nucleus accumbens, the sucrose bolus increased Glut3 mRNA and decreased Glut4 mRNA independent of prior diet exposure. The ATP-sensitive potassium channel subunit Kir6.1 in the nucleus accumbens tended to be affected by the synergistic effects of a fcHFD and a sucrose bolus. These data suggest that acute glucose handling in the hypothalamus and nucleus accumbens may be affected by prior high fat exposure.

Neuropeptide Y Signaling in the Lateral Hypothalamus Modulates Diet Component Selection and is Dysregulated in a Model of Diet-Induced Obesity.

The preclinical multicomponent free-choice high-fat high-sucrose (fcHFHS) diet has strong validity to model diet-induced obesity (DIO) and associated maladaptive molecular changes in the central nervous system. fcHFHS-induced obese rats demonstrate increased sensitivity to intracerebroventricular infusion of the orexigenic Neuropeptide Y (NPY). The brain region-specific effects of NPY signaling on fcHFHS diet component selection are not completely understood. For example, fcHFHS-fed rats have increased intake of chow and fat following intracerebroventricular NPY infusion, whereas NPY administration in the nucleus accumbens, a key hub of the reward circuitry, specifically increases fat intake. Here, we investigated whether NPY infusion in the lateral hypothalamic area (LHA), which is crucially involved in the regulation of intake, regulates fcHFHS component selection, and if LHA NPY receptor subtypes 1 or 5 (NPYR1/5) are involved. Male Wistar rats were fed a chow or fcHFHS diet for at least seven days, and received intra-LHA vehicle or NPY infusions in a cross-over design. Diet component intake was measured two hours later. Separate experimental designs were used to test the efficacy of NPY1R- or NPY5R antagonism to prevent the orexigenic effects of intra-LHA NPY. Intra-LHA NPY increased caloric intake in chow- and fcHFHS-fed rats. This effect was mediated specifically by chow intake in fcHFHS-fed rats. The orexigenic effects of intra-LHA NPY were prevented by NPY1R and NPY5R antagonism in chow-fed rats, but only by NPY5R antagonism in fcHFHS-fed rats. Thus, NPY signaling has brain region-specific effects on fcHFHS component selection and LHA NPYR sensitivity is dysregulated during consumption of a fcHFHS diet.

One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood-brain barrier permeability in fed or overnight fasted rats.

Objectives: The hypothalamus lies adjacent to the third ventricle and is in close proximity with the median eminence (ME), a circumventricular organ with an incomplete blood-brain barrier (BBB) which controls direct entry of nutrients into the brain. The blood-CSF barrier of the hypothalamus shows dynamic changes upon neuroendocrine events and adjusts permeability with the tight junction (TJ) complex. It has been shown that chronic exposure to a high-fat diet (HFD) affects BBB permeability. HFD also induces leptin resistance and alters neuropeptide expression in the arcuate nucleus (Arc) of the hypothalamus starting early during overnutrition. We hypothesized altered integrity of the BBB to occur after exposing rats to a free-choice high-fat high-sugar (fcHFHS) diet for 1 week. Methods: We measured diffusion of Evans blue dye over the ME and assessed expression of the TJ proteins ZO-1, claudin-5, and occludin in the tanycytic wall of the third ventricle. Furthermore, we assessed protein expression of glucose transporter 1 (GLUT-1), which is highly expressed in the Arc-ME complex and facilitates glucose transport over the BBB. Results: fcHFHS-fed rats increased caloric intake compared to control, however, there was no effect of the fcHFHS diet on permeability of the BBB, nor changes in protein expression of tight TJ proteins or GLUT-1. Fasting acutely affects the BBB and we hypothesized that exposure to the fcHFHS diet affects the BBB differently compared to chow after fasting. We did not, however, find any differences in Evans blue diffusion nor protein expression between chow- and fcHFHS-fed rats when fasted overnight. Conclusions: We conclude that short-term consumption of a fcHFHS diet does not change permeability or diffusion in the hypothalamus barrier in ad libitum fed or fasted rats.

TUB gene expression in hypothalamus and adipose tissue and its association with obesity in humans.

BACKGROUND/OBJECTIVES: Mutations in the Tubby gene (TUB) cause late-onset obesity and insulin resistance in mice and syndromic obesity in humans. Although TUB gene function has not yet been fully elucidated, studies in rodents indicate that TUB is involved in the hypothalamic pathways regulating food intake and adiposity. Aside from the function in central nervous system, TUB has also been implicated in energy metabolism in adipose tissue in rodents. We aimed to determine the expression and distribution patterns of TUB in man as well as its potential association with obesity. SUBJECTS/METHODS: In situ hybridization was used to localize the hypothalamic regions and cells expressing TUB mRNA. Using RT-PCR, we determined the mRNA expression level of the two TUB gene alternative splicing isoforms, the short and the long transcript variants, in the hypothalami of 12 obese and 12 normal-weight subjects, and in biopsies from visceral (VAT) and subcutaneous (SAT) adipose tissues from 53 severely obese and 24 non-obese control subjects, and correlated TUB expression with parameters of obesity and metabolic health. RESULTS: Expression of both TUB transcripts was detected in the hypothalamus, whereas only the short TUB isoform was found in both VAT and SAT. TUB mRNA was detected in several hypothalamic regions involved in body weight regulation, including the nucleus basalis of Meynert and the paraventricular, supraoptic and tuberomammillary nuclei. We found no difference in the hypothalamic TUB expression between obese and control groups, whereas the level of TUB mRNA was significantly lower in adipose tissue of obese subjects as compared to controls. Also, TUB expression was negatively correlated with indices of body weight and obesity in a fat-depot-specific manner. CONCLUSIONS: Our results indicate high expression of TUB in the hypothalamus, especially in areas involved in body weight regulation, and the correlation between TUB expression in adipose tissue and obesity. These findings suggest a role for TUB in human obesity.

Impact of obesity on taste receptor expression in extra-oral tissues: emphasis on hypothalamus and brainstem.

Sweet perception promotes food intake, whereas that of bitterness is inhibitory. Surprisingly, the expression of sweet G protein-coupled taste receptor (GPCTR) subunits (T1R2 and T1R3) and bitter GPCTRs (T2R116, T2R118, T2R138 and T2R104), as well as the α-subunits of the associated signalling complex (αGustducin, Gα14 and αTransducin), in oral and extra-oral tissues from lean and obese mice, remains poorly characterized. We focused on the impact of obesity on taste receptor expression in brain areas involved in energy homeostasis, namely the hypothalamus and brainstem. We demonstrate that many of the GPCTRs and α-subunits are co-expressed in these tissues and that obesity decreases expression of T1R3, T2R116, Gα14, αTrans and TRPM5. In vitro high levels of glucose caused a prominent down-regulation of T1R2 and Gα14 expression in cultured hypothalamic neuronal cells, leptin caused a transient down-regulation of T1R2 and T1R3 expression. Intriguingly, expression differences were also observed in other extra-oral tissues of lean and obese mice, most strikingly in the duodenum where obesity reduced the expression of most bitter and sweet receptors. In conclusion, obesity influences components of sweet and bitter taste sensing in the duodenum as well as regions of the mouse brain involved in energy homeostasis, including hypothalamus and brainstem.

Expression of 11ß-hydroxysteroid dehydrogenase type 1 in the human hypothalamus.

The hypothalamus is a major target for glucocorticoids and a key structure for hypothalamic-pituitary-adrenal (HPA) axis setpoint regulation. The enzyme 11ß hydroxysteroid dehydrogenase type 1 (11ßHSD1) modulates glucocorticoid signalling in various tissues at the prereceptor level by converting biologically inactive cortisone to its active form cortisol. The present study aimed to assess 11ßHSD1 expression in the human hypothalamus. We studied 11ßHSD1 expression in five frozen and four formalin-fixed, paraffin-embedded human hypothalami (obtained from the Netherlands Brain Bank) by the polymerase chain reaction and immunocytochemistry, respectively. 11ßHSD1 mRNA was expressed in the area of the suprachiasmatic nucleus, which is the biological clock of the brain, in the supraoptic nucleus and paraventricular nucleus (PVN), and in the infundibular nucleus, which is the human homologue of the rodent arcuate nucleus. 11ßHSD1 was detected by immunocytochemistry in the same nuclei. In the PVN, neuronal 11ßHSD1 immunoreactivity colocalised with corticotrophin-releasing hormone (CRH), arginine vasopressin and oxytocin, as shown by dual fluorescence staining. Our data demonstrate that 11ßHSD1 is widely expressed in the human hypothalamus. Its colocalisation with CRH in the PVN suggests a role in modulation of glucocorticoid feedback of the HPA axis, whereas the expression of 11ßHSD1 in additional and functionally diverse hypothalamic nuclei points to a role for the enzyme in the regulation of metabolism, appetite and circadian rhythms.

Hypothalamic vasopressin and oxytocin mRNA expression in relation to depressive state in Alzheimer's disease: a difference with major depressive disorder.

Arginine vasopressin (AVP) and oxytocin (OXT), produced in the hypothalamic paraventricular (PVN) and supraoptic nucleus (SON), are considered to be involved in the pathophysiology of major depressive disorder (MDD). The objective of this study was to determine, for the first time, the relationship between AVP and OXT gene expression and depressive state in Alzheimer's disease (AD). Post-mortem brain tissue was obtained from six control subjects, and from a prospectively studied cohort of 23 AD patients, using the DSM-IIIR and the Cornell Scale for Depression in Dementia to determine depression diagnosis and severity. The amount of AVP and OXT mRNA was determined by in situ hybridisation. AD patients did not differ from controls with respect to the amount of AVP or OXT mRNA in the PVN or SON. Also, no differences were found between depressed and nondepressed AD patients and no relationship was found between the depression severity and AVP or OXT mRNA expression. The results indicate that AVP and OXT gene expression in the PVN and SON is unchanged in depressed AD patients compared to nondepressed AD patients. This is in contrast with the enhanced AVP gene expression in MDD, suggesting a difference in pathophysiology between MDD and depression in AD.

A direct androgenic involvement in the expression of human corticotropin-releasing hormone.

We investigated the possibility of a direct action of androgens on the expression of the human corticotropin-releasing hormone (CRH), which plays a central role in the hypothalamic-pituitary-adrenal (HPA)-axis. Colocalization of CRH and nuclear/cytoplasmic androgen receptor (AR) was found in neurons of the paraventricular nucleus (PVN) in the human hypothalamus. A potential androgen-responsive element (ARE) in the human CRH promoter was subsequently analyzed with bandshifts and cotransfections in neuroblastoma cells. In the presence of testosterone, recombinant human AR bound specifically to the CRH-ARE. Expression of AR in combination with testosterone repressed CRH promoter activity through the ARE. We conclude that androgens may directly affect CRH neurons in the human PVN via AR binding to the CRH-ARE, which may have consequences for sex-specific pathogenesis of mood disorders.

Glucocorticoids decrease thyrotropin-releasing hormone messenger ribonucleic acid expression in the paraventricular nucleus of the human hypothalamus.

The way glucocorticoids affect TRH mRNA expression in the paraventricular nucleus of the hypothalamus is still unclear. In view of its relevance for Cushing's syndrome and depression, we measured TRH mRNA expression in human hypothalami obtained at autopsy by means of quantitative TRH mRNA in situ hybridization. In corticosteroid-treated subjects (n = 10), TRH mRNA hybridization signal was decreased as compared with matched control subjects (n = 10) (Mann-Whitney U test, P = 0.02). By inference, hypercortisolism as present in patients with Cushing's syndrome or major depression may contribute to lower serum TSH or symptoms of depression by lowering hypothalamic TRH expression.

Co-localization of histamine with GABA but not with galanin in the human tuberomamillary nucleus.

The presence of GABA and galanin in histaminergic neurons was previously reported in the rodent brain but whether such co-localizations also occur in the human brain was not known. We used in situ hybridization histochemistry and immunohistochemistry to study the co-localization of histamine with GABA and galanin in neurons of the tuberomamillary nucleus of adult human posterior hypothalamus. On consecutive formalin-fixed paraffin-embedded sections, co-localization was assessed using the in situ hybridization for L-histidine decarboxylase mRNA and immunocytochemistry for glutamate decarboxylase-67 kDa or galanin in the two profiles of same cell. The pattern of distribution and number of histaminergic neurons identified by in situ hybridization of the synthesizing enzyme gene transcripts were in accordance with data reported for histamine immunoreactivity. The great majority of neurons within the main divisions of the tuberomamillary nucleus containing L-histidine decarboxylase mRNA was also immunoreactive for glutamate decarboxylase-67 kDa. The range of co-localization of the two markers varied from 72% in the lateral part, to 75-87% in the medial part and 83-88% in the ventral part. In contrast, no cell containing L-histidine decarboxylase mRNA was immunoreactive for galanin. We conclude that tuberomamillary neurons in human co-express histamine with GABA but, unlike the neurons in rodents, do not express galanin, indicating that neurotransmitter co-localization patterns differ in the two species.

Alterations in arginine vasopressin neurons in the suprachiasmatic nucleus in depression.

BACKGROUND: Circadian rhythm disturbances are frequently found in depressed subjects. Although it has been presumed that these disturbances may reflect a disorder of the circadian pacemaker, this has never been established. The suprachiasmatic nucleus (SCN) is the pacemaker of the circadian timing system in mammals, and arginine vasopressin (AVP) is one of its major neuropeptides. As peptide content is often taken as a measure for activity, we hypothesized that a decreased number of AVP-immunoreactive (AVP-IR) neurons and amount of AVP-messenger RNA (mRNA) would be present in the SCN of depressed subjects. METHODS: Brains of 11 subjects suffering from major depression (8 cases) and bipolar disorder (3 cases), and of 11 controls, matched for sex, age, and clock time at death, were collected. The number of AVP-IR neurons in the SCN was determined by means of a digitizer (CalComp Inc, Reading, England). The amount of AVP-mRNA expression in the SCN was quantified with the Interaktive Bild Analyse System image analysis system (Kontron, Munich, Germany). RESULTS: In depressed subjects, the number of AVP-IR neurons in the SCN was more than one and a half times higher than in controls, while the total masked area of silver grains, as an estimate of the amount of AVP-mRNA, was about one half that of controls. CONCLUSIONS: Contrary to our hypothesis, an increase in the number of AVP-IR neurons in the SCN in depression was found, together with an expected decrease in AVP-mRNA. These findings suggest that, in depressed patients, both the synthesis and release of AVP in the SCN is reduced, resulting in an impaired functional ability. A disbalance between AVP production and transport needs further investigation in future studies.

Decreased neuropeptide Y (NPY) expression in the infundibular nucleus of patients with nonthyroidal illness.

In patients with a variety of illnesses, serum concentrations of T3 decrease without giving rise to elevated serum levels of TSH, a phenomenon known as the sick euthyroid syndrome or nonthyroidal illness (NTI). Our previous studies in postmortem brain material showed decreased thyrotropin-releasing hormone (TRH) messenger RNA (mRNA) in the paraventricular nucleus (PVN) of patients with NTI, suggesting a role for TRH cells in the persistence of low TSH levels in NTI. In the present study, we hypothesized that changes in neuropeptide Y (NPY) input from the infundibular nucleus (IFN) to TRH cells in the PVN might be a determinant of decreased TRH expression in NTI. We investigated the hypothalamus of nine patients whose endocrine status had been assessed in a serum sample taken less than 24h before death and we examined NPY expression in the IFN by means of immunocytochemistry and mRNA in situ hybridization using an image analysis system. There was a negative correlation (r = -0.88; p = 0.01) between serum leptin concentrations and total NPY mRNA in the IFN. The total amount of NPY immunoreactivity in the IFN correlated with total NPY mRNA (r = 0.69; p = 0.04). In contrast to the situation in food-deprived rodents, total NPY immunoreactivity in the IFN showed a positive correlation with total TRH mRNA in the PVN (r = 0.77; p = 0.02). The results suggest a role for decreased NPY input from the IFN in the resetting of thyroid hormone feedback on hypothalamic TRH cells in NTI.

Metabolic activity of the human ventromedial nucleus neurons in relation to sex and ageing.

The ventromedial nucleus (VMN) in animals is involved in a number of sexually dimorphic behaviors, including reproduction, and is a well-documented target for sex steroids. In rats and in lizards, it is also characterized by the presence of structural sexual dimorphisms. In the present study, we determined whether the metabolic activity of human ventromedial nucleus neurons was sex- or age-related. The size of the immunocytochemically defined Golgi apparatus (GA) and cell profiles were determined as measures for neuronal metabolic activity in 12 male and 16 female control brains sub-divided into four groups with the dividing line being the age of 50. It appeared that the size of the GA relative to cell size was 34% larger in young women (<50 years old) than in young men and was 25% larger in elderly men (> or = 50 years old) than in young men. In addition, the GA/cell size ratio correlated significantly with age in men and not in women. Our data suggest that androgens play an inhibitory role with respect to the metabolic activity of the human VMN neurons.

Multinucleated arginine-vasopressin neurons in the human supraoptic nucleus: a hallmark of pulmonary pathology.

The human supraoptic nucleus (SON) is the main production site of plasma arginine-vasopressin (AVP). The present study aimed to define the subpopulation of patients in which multinucleated SON neurons (MNN) are present. We determined the proportion of MNN in immunocytochemically defined SON AVP population and compared AVP mRNA levels in the SON of 29 patients with and without MNN. Interestingly, MNN appeared to be present in a high proportion in patients with pneumonia and other pulmonary pathologies (Pul P). The percentage of MNN in the SON of Pul P patients turned out to be age- and sex-dependent. In young women with Pul P their proportion was 10 times higher than in women without such a pathology and in young men with Pul P their frequency was 22 times higher than in other men. In those patients with the highest proportion of MNN, i.e. young females and males with Pul P, AVP mRNA expression in the SON was the lowest. In addition, young women (less than or = 50 years old) had lower AVP mRNA levels than young men and than elderly women (>50 years old). In conclusion, our study suggests that multinucleated neurons are a hallmark of Pul P in the human SON and that this phenomenon may be accompanied by lower AVP production in young subjects.

Decreased vasopressin gene expression in the biological clock of Alzheimer disease patients with and without depression.

Circadian rhythm disturbances are frequently present in Alzheimer disease (AD). In the present study, we investigated the expression of vasopressin (AVP) mRNA in the human suprachiasmatic nucleus (SCN). The in situ hybridization procedure on formalin-fixed paraffin-embedded material was improved to such a degree that we could, for the first time, visualize AVP mRNA expressing neurons in the human SCN and carry out quantitative measurements. The total amount of AVP mRNA expressed as masked silver grains in the SCN was 3 times lower in AD patients (n = 14; 2,135 +/- 597 microm2) than in age- and time-of-death-matched controls (n = 11; 6,667 +/- 1466 microm2) (p = 0.003). No significant difference was found in the amount of AVP mRNA between AD patients with depression (n = 7) and without depression (n = 7) (2,985 +/-1103 microm2 and 1,285 +/- 298 microm2, respectively; p = 0.38). In addition, the human SCN AVP mRNA expressing neurons showed a marked day-night difference in controls under 80 years of age. The amount of AVP mRNA was more than 3 times higher during the daytime (9,028 +/- 1709 microm2, n = 7) than at night (2,536 +/- 740 microm2, n = 4; p = 0.02), whereas no clear diurnal rhythm of AVP mRNA in the SCN was observed in AD patients. There was no relationship between the amount of AVP mRNA in the SCN and age at onset of dementia, duration of AD and the neuropathological changes in the cerebral cortex. These findings suggest that the neurobiological basis of the circadian rhythm disturbances that are responsible for behavioral rhythm disorders is located in the SCN. It also explains the beneficial effects of light therapy on nightly restlessness in AD patients.