Visceral adipose tissue imparts peripheral macrophage influx into the hypothalamus.

BACKGROUND: Obesity is characterized by a systemic inflammation and hypothalamic neuroinflammation. Systemic inflammation is caused by macrophages that infiltrate obese adipose tissues. We previously demonstrated that high-fat diet (HFD)-fed male mice exhibited peripheral macrophage infiltration into the hypothalamus, in addition to activation of resident microglia. Since this infiltration contributes to neuroinflammation and neuronal impairment, herein we characterize the phenotype and origin of these hypothalamic macrophages in HFD mice. METHODS: C57BL/6J mice were fed HFD (60% kcal from fat) or control diet with matching sucrose levels, for 12-16 weeks. Males and females were analyzed separately to determine sex-specific responses to HFD. Differences in hypothalamic gene expression in HFD-fed male and female mice, compared to their lean controls, in two different areas of the hypothalamus, were determined using the NanoString neuroinflammation panel. Phenotypic changes in macrophages that infiltrated the hypothalamus in HFD-fed mice were determined by analyzing cell surface markers using flow cytometry and compared to changes in macrophages from the adipose tissue and peritoneal cavity. Adipose tissue transplantation was performed to determine the source of hypothalamic macrophages. RESULTS: We determined that hypothalamic gene expression profiles demonstrate sex-specific and region-specific diet-induced changes. Sex-specific changes included larger changes in males, while region-specific changes included larger changes in the area surrounding the median eminence. Several genes were identified that may provide partial protection to female mice. We also identified diet-induced changes in macrophage migration into the hypothalamus, adipose tissue, and peritoneal cavity, specifically in males. Further, we determined that hypothalamus-infiltrating macrophages express pro-inflammatory markers and markers of metabolically activated macrophages that were identical to markers of adipose tissue macrophages in HFD-fed mice. Employing adipose tissue transplant, we demonstrate that hypothalamic macrophages can originate from the visceral adipose tissue. CONCLUSION: HFD-fed males experience higher neuroinflammation than females, likely because they accumulate more visceral fat, which provides a source of pro-inflammatory macrophages that migrate to other tissues, including the hypothalamus. Our findings may explain the male bias for neuroinflammation and the metabolic syndrome. Together, our results demonstrate a new connection between the adipose tissue and the hypothalamus in obesity that contributes to neuroinflammation and hypothalamic pathologies.

Obesity, Neuroinflammation, and Reproductive Function.

The increasing occurrence of obesity has become a significant public health concern. Individuals with obesity have higher prevalence of heart disease, stroke, osteoarthritis, diabetes, and reproductive disorders. Reproductive problems include menstrual irregularities, pregnancy complications, and infertility due to anovulation, in women, and lower testosterone and diminished sperm count, in men. In particular, women with obesity have reduced levels of both gonadotropin hormones, and, in obese men, lower testosterone is accompanied by diminished LH. Taken together, these findings indicate central dysregulation of the hypothalamic-pituitary-gonadal axis, specifically at the level of the GnRH neuron function, which is the final brain output for the regulation of reproduction. Obesity is a state of hyperinsulinemia, hyperlipidemia, hyperleptinemia, and chronic inflammation. Herein, we review recent advances in our understanding of how these metabolic and immune changes affect hypothalamic function and regulation of GnRH neurons. In the latter part, we focus on neuroinflammation as a major consequence of obesity and discuss findings that reveal that GnRH neurons are uniquely positioned to respond to inflammatory changes.

Leukemia Inhibitory Factor Represses GnRH Gene Expression via cFOS during Inflammation in Male Mice.

BACKGROUND: The mechanisms whereby neuroinflammation negatively affects neuronal function in the hypothalamus are not clear. Our previous study determined that obesity-mediated chronic inflammation elicits sex-specific impairment in reproductive function via reduction in spine density in gonadotropin-releasing hormone (GnRH) neurons. Neuroinflammation and subsequent decrease in GnRH neuron spine density was specific for male mice, while protection in females was independent of ovarian estrogens. METHODS: To examine if neuroinflammation-induced cytokines can directly regulate GnRH gene expression, herein we examined signaling pathways and mechanisms in males in vivo and in GnRH-expressing cell line, GT1-7. RESULTS: GnRH neurons express cytokine receptors, and chronic or acute neuroinflammation represses GnRH gene expression in vivo. Leukemia inhibitory factor (LIF) in particular represses GnRH expression in GT1-7 cells, while other cytokines do not. STAT3 and MAPK pathways are activated following LIF treatment, but only MAPK pathway, specifically p38α, is sufficient to repress the GnRH gene. LIF induces cFOS that represses the GnRH gene via the -1,793 site in the enhancer region. In vivo, following high-fat diet, cFOS is induced in GnRH neurons and neurons juxtaposed to the leaky blood brain barrier of the organum vasculosum of the lamina terminalis, but not in the neurons further away. CONCLUSION: Our results indicate that the increase in LIF due to neuroinflammation induces cFOS and represses the GnRH gene. Therefore, in addition to synaptic changes in GnRH neurons, neuroinflammatory cytokines directly regulate gene expression and reproductive function, and the specificity for neuronal targets may stem from the proximity to the fenestrated capillaries.

Diet-Induced Obesity Elicits Macrophage Infiltration and Reduction in Spine Density in the Hypothalami of Male but Not Female Mice.

Increasing prevalence in obesity has become a significant public concern. C57BL/6J mice are prone to diet-induced obesity (DIO) when fed high-fat diet (HFD), and develop chronic inflammation and metabolic syndrome, making them a good model to analyze mechanisms whereby obesity elicits pathologies. DIO mice demonstrated profound sex differences in response to HFD with respect to inflammation and hypothalamic function. First, we determined that males are prone to DIO, while females are resistant. Ovariectomized females, on the other hand, are susceptible to DIO, implying protection by ovarian hormones. Males, but not females, exhibit changes in hypothalamic neuropeptide expression. Surprisingly, ovariectomized females remain resistant to neuroendocrine changes, showing that ovarian hormones are not necessary for protection. Second, obese mice exhibit sex differences in DIO-induced inflammation. Microglial activation and peripheral macrophage infiltration is seen in the hypothalami of males, while females are protected from the increase in inflammatory cytokines and do not exhibit microglia morphology changes nor monocyte-derived macrophage infiltration, regardless of the presence of ovarian hormones. Strikingly, the anti-inflammatory cytokine IL-10 is increased in the hypothalami of females but not males. Third, this study posits a potential mechanism of obesity-induced impairment of hypothalamic function whereby obese males exhibit reduced levels of synaptic proteins in the hypothalamus and fewer spines in GnRH neurons, located in the areas exhibiting macrophage infiltration. Our studies suggest that inflammation-induced synaptic remodeling is potentially responsible for hypothalamic impairment that may contribute to diminished levels of gonadotropin hormones, testosterone, and sperm numbers, which we observe and corresponds to the observations in obese humans. Taken together, our data implicate neuro-immune mechanisms underlying sex-specific differences in obesity-induced impairment of the hypothalamic function with potential consequences for reproduction and fertility.

GnRH Receptor Expression and Reproductive Function Depend on JUN in GnRH Receptor‒Expressing Cells.

Gonadotropin-releasing hormone (GnRH) from the hypothalamus regulates synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gonadotropes. LH and FSH are heterodimers composed of a common α-subunit and unique ß-subunits, which provide biological specificity and are limiting components of mature hormone synthesis. Gonadotrope cells respond to GnRH via specific expression of the GnRH receptor (Gnrhr). GnRH induces the expression of gonadotropin genes and of the Gnrhr by activation of specific transcription factors. The JUN (c-Jun) transcription factor binds to AP-1 sites in the promoters of target genes and mediates induction of the FSHß gene and of the Gnrhr in gonadotrope-derived cell lines. To analyze the role of JUN in reproductive function in vivo, we generated a mouse model that lacks JUN specifically in GnRH receptor‒expressing cells (conditional JUN knockout; JUN-cKO). JUN-cKO mice displayed profound reproductive anomalies such as reduced LH levels resulting in lower gonadal steroid levels, longer estrous cycles in females, and diminished sperm numbers in males. Unexpectedly, FSH levels were unchanged in these animals, whereas Gnrhr expression in the pituitary was reduced. Steroidogenic enzyme expression was reduced in the gonads of JUN-cKO mice, likely as a consequence of reduced LH levels. GnRH receptor‒driven Cre activity was detected in the hypothalamus but not in the GnRH neuron. Female, but not male, JUN-cKO mice exhibited reduced GnRH expression. Taken together, our results demonstrate that GnRH receptor‒expression levels depend on JUN and are critical for reproductive function.

Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice.

cFOS is a pleiotropic transcription factor, which binds to the AP1 site in the promoter of target genes. In the pituitary gonadotropes, cFOS mediates induction of FSHß and GnRH receptor genes. Herein, we analyzed reproductive function in the cFOS-deficient mice to determine its role in vivo. In the pituitary cFOS is necessary for gonadotropin subunit expression, while TSHß is unaffected. Additionally, cFOS null animals have the same sex-steroid levels, although gametogenesis is impeded. In the brain, cFOS is not necessary for GnRH neuronal migration, axon targeting, cell number, or mRNA levels. Conversely, cFOS nulls, particularly females, have decreased Kiss1 neuron numbers and lower Kiss1 mRNA levels. Collectively, our novel findings suggest that cFOS plays a cell-specific role at multiple levels of the hypothalamic-pituitary-gonadal axis, affecting gonadotropes but not thyrotropes in the pituitary, and kisspeptin neurons but not GnRH neurons in the hypothalamus, thereby contributing to the overall control of reproduction.

TALE homeodomain proteins regulate gonadotropin-releasing hormone gene expression independently and via interactions with Oct-1.

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function. Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus. Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter. Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line. In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors. Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1. We show that heterodimers containing Pbx/Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site. Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells. Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression. Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo.