Changes In Hypothalamic Expression Research Articles

OR04-06 Mkrn3, Dlk1 And Mecp2 Have Distinct Hypothalamic Expression Patterns Across Pubertal Maturation In Male And Female Mice

Abstract Disclosure: F.R. Tinano: None. S.A. Pereira: None. N.P. Boris: None. D.B. Macedo: None. A.P. Canton: None. A. Abreu: None. R.S. Carroll: None. A. Latronico: None. U.B. Kaiser: None. Context: Central precocious puberty (CPP) results from premature re-activation of the hypothalamic-pituitary-gonadal (HPG) axis. The most prevalent genetic cause of CPP is loss-of-function mutations in makorin ring finger 3 (MKRN3), with loss-of-function mutations in delta-like 1 homolog (DLK1) occurring less frequently. While the mechanisms by which MKRN3 regulates puberty onset have been partially elucidated in recent years, the manner by which DLK1 regulates HPG axis maturation remains unknown. Methyl CpG binding protein 2 (MeCP2) is associated with the neurodevelopmental Rett syndrome, with reports of early pubertal development among affected patients. Recently, rare variants in MeCP2 were identified in children with CPP with or without neurologic abnormalities. The pathways by which MECP2 may influence pubertal timing have not been determined. The aim of this study was to characterize Mkrn3, Dlk1 and Mecp2 expression patterns in the hypothalamus of male and female mice at different pubertal stages.Methods and Results: Male and female wild type C57BL/6 mice were euthanized at postnatal (P) age 10, 15, 22, 30 and 60 days. The mediobasal hypothalamus (MBH) and pre-optic area (POA) were isolated from the brains of each mouse. mRNA levels of Mkrn3, Dlk1 and Mecp2 were quantified by RT-qPCR and compared across the ages studied. Mkrn3 expression in both MBH and POA was highest at P10, then significantly lower at P15 and P22, remaining low until adulthood, with a similar pattern in male and female mice. In contrast, Dlk1 expression was higher at P60 compared to P22 in the MBH of male mice, while in female mice it was higher at P22 and P30 compared to P10. In the POA of male mice, Dlk1 expression was higher at P30 and P60 compared to P10, while no significant difference with age was seen in the POA of female mice. Mecp2 had higher expression in the MBH at P10 compared to P15 and P22 in male mice, and compared to P15, P30 and P60 in female mice. Mecp2 expression in the POA of both male and female mice was higher at P10 compared to all the other ages. No differences were observed in the hypothalamic expression of the three genes between male and female mice. Statistical significance was set at p < 0.05. Conclusions:Mkrn3 and Mecp2 had decreased hypothalamic expression in male and female mice with age, suggesting inhibitory roles on the HPG axis, consistent with the phenotype of CPP in patients with loss-of-function mutations in these genes. In contrast, hypothalamic expression of Dlk1 increased in both male and female mice toward adulthood, which is intriguing considering the association of loss-of-function mutations in this gene with CPP and suggests that the role of Dlk1 in the HPG axis is distinct from those of Mkrn3 and Mecp2. Defining the changes in hypothalamic expression of these three genes across pubertal development is critical to understanding their roles and mechanisms of action in regulating the HPG axis. Presentation: Thursday, June 15, 2023

Effects of incubation lighting with green or white light on brown layers: hatching performance, feather pecking and hypothalamic expressions of genes related with photoreception, serotonin, and stress systems

The aim of this study was to evaluate the effect of 16L:8D photoperiod with green (GREEN) or white (WHITE) lights during incubation on hatching performance, blood melatonin, corticosterone, and serotonin levels, hypothalamic expressions of genes related to photoreception, serotonin, and stress systems in layers in relation with feather pecking behavior. Dark incubation (DARK) was the control. Eggs (n = 1,176) from Brown Nick breeders in 2 batches (n = 588/batch) were incubated in the experiment. A total of 396 female chicks and 261 hens were used at rearing and laying periods until 40 wk. Incubation lighting did not affect hatchability, day-old chick weight, and length, but resulted in a more synchronized hatch as compared with the DARK. The effect of incubation lighting on blood hormones was not significant except for reduced serotonin in the GREEN group at the end of the experiment. There was no effect of incubation lighting on gentle, severe, and aggressive pecking of birds during the early rearing period. From 16 wk, GREEN hens showed increased gentle pecking with increasing age. WHITE hens had the highest gentle pecking frequency at 16 wk while they performed less gentle but higher severe and aggressive pecks at 24 and 32 wk. At hatching, the hypothalamic expression of CRH, 5-HTR1A, and 5-HTR1B was higher for the WHITE group compared with both GREEN and DARK, however, 5-HTT expression was higher in GREEN than WHITE which was similar to DARK. Except for the highest VA opsin expression obtained for WHITE hens at 40 wk of age, there was no change in hypothalamic expression levels of rhodopsin, VA opsin, red, and green opsins at any age. Although blood hormone levels were not consistent, results provide preliminary evidence that incubation lighting modulates the pecking tendencies of laying hens, probably through the observed changes in hypothalamic expression of genes related to the serotonin system and stress. Significant correlations among the hypothalamic gene expression levels supplied further evidence for the associations among photoreception, serotonin, and stress systems.

The hypophagic response to heat stress is not mediated by GPR109A or peripheral β-OH butyrate

Rising temperatures resulting from climate change will increase the incidence of heat stress, negatively impacting the labor force and food animal production. Heat stress elevates circulating β-OH butyrate, which induces vasodilation through GPR109a. Interestingly, both heat stress and intraperitoneal β-OH butyrate administration induce hypophagia. Thus, we aimed to investigate the role of β-OH butyrate in heat stress hypophagia in mice. We found that niacin, a β-OH butyrate mimetic that cannot be oxidized to generate ATP, also reduces food intake. Interestingly, the depression in food intake as a result of 8-h intraperitoneal niacin or 48-h heat exposure did not result from changes in hypothalamic expression of orexigenic or anorexigenic signals (AgRP, NPY, or POMC). Genetically eliminating GPR109a expression did not prevent the hypophagic response to heat exposure, intraperitoneal β-OH butyrate (5.7 mmol/kg), or niacin (0.8 mmol/kg). Hepatic vagotomy eliminated the hypophagic response to β-OH butyrate and niacin but did not affect the hypophagic response to heat exposure. We subsequently hypothesized that the hypophagic response to heat stress may depend on direct effects of β-OH butyrate at the central nervous system: β-OH butyrate induced hormonal changes (hyperinsulinemia, hypercorticosteronemia, and hyperleptinemia), or gene expression changes. To test these possibilities, we blocked expression of hepatic hydroxyl methyl glutaryl CoA synthase II (HMGCS2) to prevent hepatic β-OH butyrate synthesis. Mice that lack HMGCS2 maintain a hypophagic response to heat stress. Herein, we establish that the hypophagia of heat stress is independent of GPR109a, the hepatic vagus afferent nerve, and hepatic ketone body synthesis.

Prenatal undernutrition results in greater lipopolysaccharide-induced changes in hypothalamic TNF-α expression, but does not affect the equivalent changes in the serum levels of luteinizing hormone and testosterone, in adult male rats

Immune stress can cause reproductive dysfunction. Some hypothalamic factors such as pro-inflammatory cytokines play pivotal roles in reproductive disorders under immune stress conditions. Recently, it has been reported that prenatal undernutrition affects not only metabolic functions, but also the responses of physiological functions to immune stress in adulthood. In this study, the long-term effects of prenatal undernutrition on the responses of hypothalamic pro-inflammatory cytokine (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6) expression; reproductive endocrine factors; i.e., the serum levels of gonadotropins and testosterone; and hypothalamic kisspeptin expression to lipopolysaccharide (LPS) were examined in male rats. Pregnant rats were divided into two groups; i.e., the normally nourished group and the undernourished (50% food restricted) group. The offspring of the normally nourished mothers (control) and undernourished mothers (the intrauterine growth restriction [IUGR] group) were sub-divided into saline-injected and LPS (500μg, i.p.)-injected groups at 10 weeks of age. The rats’ hypothalamic pro-inflammatory cytokine levels and serum luteinizing hormone (LH) and testosterone levels were measured and compared between the control and IUGR groups. The hypothalamic pro-inflammatory cytokine mRNA levels of the LPS-injected rats were significantly higher than those of the saline-injected rats in both the control and IUGR groups. The changes in the hypothalamic expression level of TNF-α, but not those of the other cytokines, induced in response to LPS were more marked in the IUGR group than in the control group. On the other hand, although the serum LH and testosterone levels of the LPS-injected rats were significantly lower than those of the saline-injected rats in both the control and IUGR groups, their levels did not differ between the control and IUGR groups under the LPS-injected conditions. These results suggest that prenatal undernutrition results in more marked LPS-induced changes in hypothalamic TNF-α expression, but does not alter the effects of LPS on the serum levels of LH or testosterone, in adult male rats.

KISS1 gene expression in the developing brain of female pigs in pre- and peripubertal periods.

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P<0.05), suggesting a developing negative feedback mechanism affecting gonadotropin release during the prepubertal period. Considering the potent stimulating effect of kisspeptin on gonadotropin release in prepubertal pigs, kisspeptin secretion rather than kisspeptin synthesis may be responsible for the onset of puberty in pigs.

Changes in Hypothalamic Expression of the Lin28/let-7 System and Related MicroRNAs During Postnatal Maturation and After Experimental Manipulations of Puberty

Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.

Herpes Virus Entry Mediator Signaling in the Brain Is Imperative in Acute Inflammation-Induced Anorexia and Body Weight Loss

BackgroundReduced appetite and body weight loss are typical symptoms of inflammatory diseases. A number of inflammatory stimuli are responsible for the imbalance in energy homeostasis, leading to metabolic disorders. The herpes virus entry mediator (HVEM) protein plays an important role in the development of various inflammatory diseases, such as intestinal inflammation and diet-induced obesity. However, the role of HVEM in the brain is largely unknown. This study aims to investigate whether HVEM signaling in the brain is involved in inflammation-induced anorexia and body weight loss.MethodsFood intake and body weight were measured at 24 hours after intraperitoneal injection of lipopolysaccharide (LPS) or intracerebroventricular injection of recombinant mouse LIGHT (also called tumor necrosis factor receptor superfamily 14, TNFSF14), an HVEM ligand, into 8- to 10-week-old male C57BL/6 mice and mice lacking HVEM expression (HVEM-/-). We also assessed LPS-induced change in hypothalamic expression of HVEM using immunohistochemistry.ResultsAdministration of LPS significantly reduced food intake and body weight, and moreover, increased expression of HVEM in the hypothalamic arcuate nucleus. However, LPS induced only minor decreases in food intake and body weight in HVEM-/- mice. Administration of LIGHT into the brain was very effective at decreasing food intake and body weight in wild-type mice, but was less effective in HVEM-/- mice.ConclusionActivation of brain HVEM signaling is responsible for inflammation-induced anorexia and body weight loss.

Functional requirement of AgRP and NPY neurons in ovarian cycle-dependent regulation of food intake

In female mammals including rodents and humans, feeding decreases during the periovulatory period of the ovarian cycle, which coincides with a surge in circulating estrogen levels. Ovariectomy increases food intake, which can be normalized by estrogen treatment at a dose and frequency mimicking those during the estrous cycle. Furthermore, administration of estrogen to rodents potently inhibits food intake. Despite these well-known effects of estrogen, neuronal subtypes that mediate estrogen's anorexigenic effects have not been identified. In this study, we show that changes in hypothalamic expression of agouti-related protein (Agrp) and neuropeptide Y (Npy) coincide with the cyclic changes in feeding across the estrous cycle. These cyclic changes in feeding are abolished in mice with degenerated AgRP neurons even though these mice cycle normally. Central administration of 17beta-estradiol (E2) decreases food intake in controls but not in mice lacking the AgRP neurons. Furthermore, E2 treatment suppresses fasting-induced c-Fos activation in AgRP and NPY neurons and blunts the refeeding response. Surprisingly, although estrogen receptor alpha (ERalpha) is the key mediator of estrogen's anorexigenic effects, we find that expression of ERalpha is completely excluded from AgRP and NPY neurons in the mouse hypothalamus, suggesting that estrogen may regulate these neurons indirectly via presynaptic neurons that express ERalpha. This study indicates that neurons coexpressing AgRP and NPY are functionally required for the cyclic changes in feeding across estrous cycle and that AgRP and NPY neurons are essential mediators of estrogen's anorexigenic function.

Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence fertility are yet to be completely deciphered. Recently, the hypothalamic KiSS-1/GPR54 system has emerged as a fundamental regulator of the gonadotropic axis, which conveys the modulatory actions of sex steroids to GnRH neurons. Evidence is also mounting that KiSS-1 neurons may also represent the link between systemic metabolic signals and central control of reproduction. To further explore this possibility, we examined the impact of changes in energy status and key metabolic regulators on the hypothalamic expression of KiSS-1 and GPR54 genes, using different mouse models and the hypothalamic cell line N6. Time-course analysis of the effects of short-term fasting revealed a rapid (12- and 24-h) decline in KiSS-1 and GPR54 mRNA levels, which preceded that of GnRH (48 h). In contrast, diet-induced obesity or obesity associated with leptin deficiency (ob/ob vs. wild-type mice) failed to induce overt changes in hypothalamic expression of KiSS-1 and GPR54 genes. However, leptin infusion of ob/ob mice evoked a significant increase in KiSS-1 and GPR54 mRNA levels compared with pair-fed controls. Moreover, leptin, but not insulin or IGF-I, stimulated KiSS-1 mRNA expression in the mouse hypothalamic cell line N6. In addition, neuropeptide Y (NPY) null mice showed decreased KiSS-1 mRNA levels at the hypothalamus, whereas exposure to NPY increased expression of KiSS-1 in hypothalamic N6 cells. In sum, our present data further characterize the functional relevance and putative key mediators (such as leptin and NPY) of the metabolic regulation of the hypothalamic KiSS-1 system in the mouse.

Appetite suppressing effect of t10, c12 conjugated linoleic acid on mice is dependent on dietary fat level and the temporal pattern of energy intake is associated with changes in hypothalamic expression of genes involved in appetite control.

Appetite suppressing effect of t10, c12 conjugated linoleic acid on mice is dependent on dietary fat level and the temporal pattern of energy intake is associated with changes in hypothalamic expression of genes involved in appetite control.

Hypothalamic Expression of KiSS-1 System and Gonadotropin-Releasing Effects of Kisspeptin in Different Reproductive States of the Female Rat

Kisspeptins, products of the KiSS-1 gene with ability to bind G protein-coupled receptor 54 (GPR54), have been recently identified as major gatekeepers of reproductive function with ability to potently activate the GnRH/LH axis. Yet, despite the diversity of functional states of the female gonadotropic axis, pharmacological characterization of this effect has been mostly conducted in pubertal animals or adult male rodents, whereas similar studies have not been thoroughly conducted in the adult female. In this work, we evaluated maximal LH and FSH secretory responses to kisspeptin-10, as well as changes in sensitivity and hypothalamic expression of KiSS-1 and GPR54 genes, in different physiological and experimental models in the adult female rat. Kisspeptin-10 (1 nmol, intracerebroventricular) was able to elicit robust LH bursts at all phases of the estrous cycle, with maximal responses at estrus; yet, in diestrus LH, responses to kisspeptin were detected at doses as low as 0.1 pmol. In contrast, high doses of kisspeptin only stimulated FSH secretion at diestrus. Removal of ovarian sex steroids did not blunt the ability of kisspeptin to further elicit stimulated LH and FSH secretion, but restoration of maximal responses required replacement with estradiol and progesterone. Finally, despite suppressed basal levels, LH and FSH secretory responses to kisspeptin were preserved in pregnant and lactating females, although the magnitude of LH bursts and the sensitivity to kisspeptin were much higher in pregnant dams. Interestingly, hypothalamic KiSS-1 gene expression significantly increased during pregnancy, whereas GPR54 mRNA levels remained unaltered. In summary, our current data document for the first time the changes in hypothalamic expression of KiSS-1 system and the gonadotropic effects (maximal responses and sensitivity) of kisspeptin in different functional states of the female reproductive axis. The present data may pose interesting implications in light of the potential therapeutic use of kisspeptin analogs in the pharmacological manipulation of the gonadotropic axis in the female.

Impact of Obesity on the Growth Hormone Axis: Evidence for a Direct Inhibitory Effect of Hyperinsulinemia on Pituitary Function

There is a negative relationship between obesity and GH. However, it is not known how metabolic changes, associated with obesity, lead to a reduction in GH output. This study examined the GH axis of two mouse models of obesity, the leptin-deficient (ob/ob) mouse and the diet-induced obese (DIO; high-fat fed) mouse. Both models displayed hyperglycemia and hyperinsulinemia with reduced expression of GH as well as reduced expression of pituitary receptors important for GH synthesis and release [GHRH receptor (DIO only) and the ghrelin receptor (ob/ob and DIO)]. These pituitary changes were not accompanied by changes in hypothalamic expression of GHRH or somatostatin; suggesting that alterations in pituitary function may be precipitated in part by direct effects of systemic signals. Of the metabolic and hormonal parameters examined (insulin, glucose, corticosterone, free fatty acids, ghrelin, and IGF-I), only insulin/glucose showed a significant, and negative, correlation with pituitary expression. Pituitaries of DIO mice remained responsive to the acute in vivo actions of insulin, as assessed by phosphorylation of Akt, despite systemic (skeletal muscle and fat) insulin resistance. In addition, treating primary pituitary cell cultures from lean mice with insulin reduced GH release as well as GH, GHRH receptor, and ghrelin receptor mRNA levels compared with vehicle-treated controls, where the magnitude of suppression of pituitary mRNA levels was similar to that observed in the DIO mouse. These results coupled with the fact that the pituitary expresses the insulin receptor at levels comparable to tissues classically considered insulin sensitive, indicates high circulating insulin levels can directly contribute to the suppression of GH synthesis and release in the obese state.

Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides.

To investigate the mechanisms underlying long-term resistance of the A/J mouse strain to diet-induced obesity, we studied, over a period of 4 wk, the expression of uncoupling proteins in brown adipose tissue and the expression of hypothalamic neuropeptides known to regulate energy homeostasis and then used microarray analysis to identify other potentially important hypothalamic peptides. Despite increased caloric intake after 2 days of high-fat feeding, body weights of A/J mice remained stable. On and after 1 wk of high-fat feeding, A/J mice adjusted their food intake to consume the same amount of calories as mice fed a low-fat diet; thus their body weight and insulin, corticosterone, free fatty acid, and glucose levels remained unchanged for 4 wk. We found no changes in hypothalamic expression of several orexigenic and/or anorexigenic neuropeptides known to play an important role in energy homeostasis for the duration of the study. Uncoupling protein-2 mRNA expression in brown adipose tissue, however, was significantly upregulated after 2 days of high-fat feeding and tended to remain elevated for the duration of the 4-wk study. Gene array analysis revealed that several genes are up- or downregulated in response to 2 days and 1 wk of high-fat feeding. Real-time PCR analysis confirmed that expression of the hypothalamic IL-1 pathway (IL-1beta, IL-1 type 1 and 2 receptors, and PPM1b/PP2C-beta, a molecule that has been implicated in the inhibition of transforming growth factor-beta-activated kinase-1-mediated IL-1 action) is altered after 2 days, but not 1 wk, of high-fat feeding. The role of additional molecules discovered by microarray analysis needs to be further explored in the future.