α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors.

Emerging evidence suggests that new cells, including neurons, can be generated within the adult hypothalamus, suggesting the existence of a local neural stem/progenitor cell niche. Here, we identify α-tanycytes as key components of a hypothalamic niche in the adult mouse. Long-term lineage tracing in vivo using a GLAST::CreER(T2) conditional driver indicates that α-tanycytes are self-renewing cells that constitutively give rise to new tanycytes, astrocytes and sparse numbers of neurons. In vitro studies demonstrate that α-tanycytes, but not ß-tanycytes or parenchymal cells, are neurospherogenic. Distinct subpopulations of α-tanycytes exist, amongst which only GFAP-positive dorsal α2-tanycytes possess stem-like neurospherogenic activity. Fgf-10 and Fgf-18 are expressed specifically within ventral tanycyte subpopulations; α-tanycytes require fibroblast growth factor signalling to maintain their proliferation ex vivo and elevated fibroblast growth factor levels lead to enhanced proliferation of α-tanycytes in vivo. Our results suggest that α-tanycytes form the critical component of a hypothalamic stem cell niche, and that local fibroblast growth factor signalling governs their proliferation.

The antioxidant anethole dithiolethione inhibits monoamine oxidase-B but not monoamine oxidase A activity in extracts of cultured astrocytes.

Anethole dithiolethione (ADT) is a clinically available, pluripotent antioxidant proposed as a neuroprotectant for Parkinson's disease (PD). Here, using extracts from cultured astrocytes, containing both monoamine oxidase (MAO) A and B activity, we demonstrate that ADT concentration-dependently inhibits MAO-B activity in a clinically relevant concentration range (0.03-30 microM, IC-50 = 0.5 microM) without affecting MAO A activity. Considering the alleged contribution of MAO activity in general, and MAO-B in particular, to oxidative stress and neurodegeneration in PD, our data further support the neuroprotective potential of ADT.

A transgenic model of visceral obesity and the metabolic syndrome.

The adverse metabolic consequences of obesity are best predicted by the quantity of visceral fat. Excess glucocorticoids produce visceral obesity and diabetes, but circulating glucocorticoid levels are normal in typical obesity. Glucocorticoids can be produced locally from inactive 11-keto forms through the enzyme 11beta hydroxysteroid dehydrogenase type 1 (11beta HSD-1). We created transgenic mice overexpressing 11beta HSD-1 selectively in adipose tissue to an extent similar to that found in adipose tissue from obese humans. These mice had increased adipose levels of corticosterone and developed visceral obesity that was exaggerated by a high-fat diet. The mice also exhibited pronounced insulin-resistant diabetes, hyperlipidemia, and, surprisingly, hyperphagia despite hyperleptinemia. Increased adipocyte 11beta HSD-1 activity may be a common molecular etiology for visceral obesity and the metabolic syndrome.

Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation.

Recruitment of activated T-cells to the skin is a common feature in a wide variety of inflammatory skin diseases. As CXCR3 activating chemokines CXCL10 (IP-10), CXCL9 (Mig), and CXCL11 (IP-9/I-TAC) specifically attract activated T-cells, this study addressed the question of whether differences in the expression of these chemokines correlate with the site and cellular composition of the skin infiltrates in different types of inflammatory skin disease. Skin biopsies from lichen planus, chronic discoid lupus erythematosus, allergic patch test reactions, psoriasis, and Jessner's lymphocytic infiltration of the skin were investigated for chemokine expression using RNA in situ hybridization, and for the expression of CXCR3 using immunohistochemistry. The results showed differential expression of CXCL10, CXCL9, and CXCL11, which correlated with differences in the localization and cellular composition of the infiltrates. Whereas CXCL10 and CXCL11 were mainly expressed by basal keratinoctyes, CXCL9 mRNA expression was located predominantly in the dermal infiltrates. Correlation with immunohistochemical data suggested that macrophages and activated keratinocytes were the main producers of these chemokines. CXCR3 was expressed by a majority of both CD4+ and CD8+ infiltrating T-cells, suggesting a functional interaction between locally produced chemokines and CXCR3-expressing T-cells. In conclusion, these findings indicate that these CXCR3 activating chemokines play a significant role in the recruitment and maintenance of T-cell infiltrates in the inflammatory skin diseases studied.

Synchronicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: evidence for possible partial TSH regulation by leptin in humans.

Leptin signals the status of energy reserves to the brain. Leptin stimulates biosynthesis of TRH in vitro and influences the activity of the hypothalamic-pituitary-thyroid axis in vivo in rodents. Because blood levels of both leptin and TSH display diurnal variation with a distinct nocturnal rise, we sought to determine whether a relationship exists between fluctuations in circulating leptin and TSH. We measured serum leptin and TSH levels every 7 min for 24 h in five healthy men and found that both leptin and TSH levels are highly organized and pulsatile. A similar pattern of leptin and TSH rhythms was observed, with TSH and leptin levels reaching a nadir in late morning and a peak in the early morning hours. Importantly, cosinor analysis on the absolute leptin and TSH levels revealed a statistically significant fit for a 24-h period and the two hormones showed similar probabilities of rhythm and superimposable peak values. Furthermore, this study shows a strong positive Pearson correlation between the 24-h patterns of variability of leptin and TSH in healthy subjects. Finally, the ultradian fluctuations in leptin levels showed pattern synchrony with those of TSH as determined by cross-correlation analysis, by cross-approximate enthropy and Bayessian analysis applied independently. To further explore whether these associations could reflect an underlying regulation of TSH secretion by leptin, we also studied frequently sampled leptin and TSH levels in four brothers, members of a family with leptin deficiency (one normal homozygote, two heterozygotes, and one leptin-deficient homozygote). Leptin levels of the homozygous leptin-deficient subject are detectable but bioinactive, and the rhythm of his TSH is disorganized. 24-h pattern of leptin and TSH variability in the heterozygous subjects, although significantly correlated, showed a weaker correlation compared with the strong correlation in the normal subjects. These data are consistent with the possibility that leptin may regulate TSH pulsatility and circadian rhythmicity, but interventional studies are needed to definitively prove whether leptin regulates the minute-to-minute oscillations and ultradian rhythm of TSH levels.

The antipsoriatic drug dimethylfumarate strongly suppresses chemokine production in human keratinocytes and peripheral blood mononuclear cells.

BACKGROUND: The effectiveness of systemic treatment of psoriasis with fumaric acid esters has been proven, but their mode of action at the cellular and molecular level has not yet been fully elucidated. OBJECTIVES: To study the effect of dimethylfumarate (DMF) on the production of the chemokines CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11, formerly known as GROalpha, interleukin-8, Mig, IP-10 and IP-9/I-TAC, respectively, in human keratinocytes and peripheral blood mononuclear cells (PBMC). METHODS: Cultured keratinocytes were stimulated with interferon (IFN) -gamma to produce CXCL9, CXCL10 and CXCL11 and with phorbol myristate acetate to produce CXCL1 and CXCL8 in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). PBMC were stimulated with either IFN-gamma to produce CXCL9 and CXCL10 or lipopolysaccharide to produce CXCL8, in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). RNA preparations from isolated keratinocytes were analysed by Northern blotting; protein production by keratinocytes and PBMC was monitored by an enzyme-linked immunosorbent assay. RESULTS: Northern blot analysis on isolated keratinocyte RNA preparations showed a dose-dependent inhibition of CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11 transcription by DMF. At 45 micromol L(-1) the inhibition was almost complete. In addition, keratinocytes and PBMC showed in the presence of DMF a dose-dependent inhibition of CXCL8, CXCL9 and CXCL10 protein production. CONCLUSIONS: These results show the ability of DMF to inhibit the production of chemokines that may be critically involved in the development and perpetuation of psoriatic lesions. This might explain, at least in part, the beneficial effects of treatment with fumaric acid esters in psoriasis patients.

Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance.

Several lines of investigation suggest that the hypothalamic neuropeptide melanin-concentrating hormone (MCH) regulates body weight in mammals. Obese mice lacking functional leptin overexpress the MCH message in the fed or fasted state. Acute intracerebroventricular injection of MCH increases energy intake in rats. Mice lacking the MCH gene are lean. To test the hypothesis that chronic overexpression of MCH in mice causes obesity, we produced transgenic mice that overexpress MCH (MCH-OE) in the lateral hypothalamus at approximately twofold higher levels than normal mice. On the FVB genetic background, homozygous transgenic animals fed a high-fat diet ate 10% more and were 12% heavier at 13 weeks of age than wild-type animals, and they had higher systemic leptin levels. Blood glucose levels were higher both preprandially and after an intraperitoneal glucose injection. MCH-OE animals were insulin-resistant, as demonstrated by markedly higher plasma insulin levels and a blunted response to insulin; MCH-OE animals had only a 5% decrease in blood glucose after insulin administration, compared with a 31% decrease in wild-type animals. MCH-OE animals also exhibited a twofold increase in islet size. To evaluate the contribution of genetic background to the predisposition to obesity seen in MCH-OE mice, the transgene was bred onto the C57BL/6J background. Heterozygote C57BL/6J mice expressing the transgene showed increased body weight on a standard diet, confirming that MCH overexpression can lead to obesity.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling.

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptin's effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.

Divergent roles of SHP-2 in ERK activation by leptin receptors.

The protein tyrosine phosphatase SHP-2 has been proposed to serve as a regulator of leptin signaling, but its specific roles are not fully examined. To directly investigate the role of SHP-2, we employed dominant negative strategies in transfected cells. We show that a catalytically inactive mutant of SHP-2 blocks leptin-stimulated ERK phosphorylation by the long leptin receptor, ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially inhibits ERK phosphorylation. We find similar effects of the SHP-2 mutants after examining stimulation of an ERK-dependent egr-1 promoter-construct by leptin. We also demonstrate ERK phosphorylation and egr-1 mRNA expression in the hypothalamus by leptin. Analysis of signaling by ObRb lacking intracellular tyrosine residues or by the short leptin receptor, ObRa, enabled us to conclude that two pathways are critical for ERK activation. One pathway does not require the intracellular domain of ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is required for both pathways, whereas activation of ERK via Tyr-985 of ObRb also requires tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator of ERK by leptin receptors, and both the adaptor function and the phosphatase activity of SHP-2 are critical for this regulation.

SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985.

During leptin signaling, each of the phosphorylated tyrosine residues on the long form of the leptin receptor (LRb) mediates distinct signals. Phosphorylated Tyr(1138) binds STAT3 to mediate its tyrosine phosphorylation and transcriptional activation, while phosphorylated Tyr(985) binds the tyrosine phosphatase SHP-2 and reportedly mediates both activation of ERK kinases and inhibition of LRb-mediated STAT3 activation. We show here that although mutation of Tyr(985) does not alter STAT3 signaling by erythropoietin receptor-LRb (ELR) chimeras in transfected 293 cells at short times of stimulation, this mutation enhances STAT3 signaling at longer times of stimulation (>6 h). These data suggest that Tyr(985) may mediate feedback inhibition of LRb signaling by an LRb-induced LRb inhibitor, such as SOCS3. Indeed, SOCS3 binds specifically to phosphorylated Tyr(985) of LRb, and SOCS3 fails to inhibit transcription by ELR following mutation of Tyr(985), suggesting that SOCS3 inhibits LRb signaling by binding to phosphorylated Tyr(985). Additionally, overexpression of SOCS3, but not SHP-2, impairs ELR signaling, and the overexpression of SHP-2 blunts SOCS3-mediated inhibition of ELR signaling. Thus, our data suggest that in addition to mediating SHP-2 binding and ERK activation during acute stimulation, Tyr(985) of LRb mediates feedback inhibition of LRb signaling by binding to LRb-induced SOCS3.

Adipose tissue as an endocrine organ.

The discovery of leptin in the mid-1990s has focused attention on the role of proteins secreted by adipose tissue. Leptin has profound effects on appetite and energy balance, and is also involved in the regulation of neuroendocrine and immune function. Sex steroid and glucocorticoid metabolism in adipose tissue has been implicated as a determinant of body fat distribution and cardiovascular risk. Other adipose products, for example, proinflammatory cytokines, complement factors and components of the coagulation/fibrinolytic cascade, may mediate the metabolic and cardiovascular complications associated with obesity.

Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice.

Exposure to high-fat diets for prolonged periods results in positive energy balance and obesity, but little is known about the initial physiological and neuroendocrine response of obesity-susceptible strains to high-fat feeding. To assess responses of C57BL/6J mice to high- and low-fat diets, we quantitated the hypothalamic expression of neuropeptides implicated in weight regulation and neuroendocrine function over a 2-wk period. Exposure to high-fat diet increased food consumption over a 2-day period during which leptin levels were increased when assessed by a frequent sampling protocol [area under the curve (AUC): 134.6 +/- 10.3 vs. 100 +/- 12.3, P = 0.03 during first day and 126.5 +/- 8.2 vs. 100 +/- 5.2, P = 0.02 during second day]. During this period, hypothalamic expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased by approximately 30 and 50%, respectively (P < 0.001). After 1 wk, both caloric intake and hypothalamic expression of NPY and AgRP returned toward baseline. After 2 wk, cumulative caloric intake was again higher in the high-fat group, and now proopiomelanocortin (POMC) was elevated by 76% (P = 0.01). This study demonstrates that high-fat feeding induces hyperphagia, hyperleptinemia, and transient suppression of orexigenic neuropeptides during the first 2 days of diet. The subsequent induction of POMC may be a second defense against obesity. Attempts to understand the hypothalamic response to high-fat feeding must examine the changes as they develop over time.

Leptin regulates prothyrotropin-releasing hormone biosynthesis. Evidence for direct and indirect pathways.

The hypothalamic-pituitary-thyroid axis is down-regulated during starvation, and falling levels of leptin are a critical signal for this adaptation, acting to suppress preprothyrotropin-releasing hormone (prepro-TRH) mRNA expression in the paraventricular nucleus of the hypothalamus. This study addresses the mechanism for this regulation, using primary cultures of fetal rat hypothalamic neurons as a model system. Leptin dose-dependently stimulated a 10-fold increase in pro-TRH biosynthesis, with a maximum response at 10 nm. TRH release was quantified using immunoprecipitation, followed by isoelectric focusing gel electrophoresis and specific TRH radioimmunoassay. Leptin stimulated TRH release by 7-fold. Immunocytochemistry revealed that a substantial population of cells expressed TRH or leptin receptors and that 8-13% of those expressing leptin receptors coexpressed TRH. Leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter and the long form of the leptin receptor cDNA. Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, addition of alpha-melanocyte-stimulating hormone produced a 3.5-fold increase in TRH biosynthesis and release, whereas neuropeptide Y treatment suppressed pro-TRH biosynthesis approximately 3-fold. Furthermore, the melanocortin-4 receptor antagonist SHU9119 partially inhibited leptin-stimulated TRH release from the neuronal culture. Consequently, our data suggest that leptin regulates the TRH neurons through both direct and indirect pathways.

Leptin regulation of neuroendocrine systems.

The discovery of leptin has enhanced understanding of the interrelationship between adipose energy stores and neuronal circuits in the brain involved in energy balance and regulation of the neuroendocrine axis. Leptin levels are dependent on the status of fat stores as well as changes in energy balance as a result of fasting and overfeeding. Although leptin was initially thought to serve mainly as an anti-satiety hormone, recent studies have shown that it mediates the adaptation to fasting. Furthermore, leptin has been implicated in the regulation of the reproductive, thyroid, growth hormone, and adrenal axes, independent of its role in energy balance. Although it is widely known that leptin acts on hypothalamic neuronal targets to regulate energy balance and neuroendocrine function, the specific neuronal populations mediating leptin action on feeding behavior and autonomic and neuroendocrine function are not well understood. In this review, we have discussed how leptin engages arcuate hypothalamic neurons expressing putative orexigenic peptides, e.g., neuropeptide Y and agouti-regulated peptide, and anorexigenic peptides, e.g., pro-opiomelanocortin (precursor of alpha-melanocyte-stimulating hormone) and cocaine- and amphetamine-regulated transcript. We show that leptin's effects on energy balance and the neuroendocrine axis are mediated by projections to other hypothalamic nuclei, e.g., paraventricular, lateral, and perifornical areas, as well as other sites in the brainstem, spinal cord, and cortical and subcortical regions.

Transcellular transport of leptin by the short leptin receptor isoform ObRa in Madin-Darby Canine Kidney cells.

Leptin is an adipocyte-derived hormone that acts in specific regions of the brain to regulate body weight and neuroendocrine function. The mechanism by which leptin enters the brain is unknown. We previously reported that rat brain microvessels, which constitute the blood-brain barrier, contain large amounts of messenger RNA encoding a short form of the leptin receptor (ObRa), suggesting that this site may be important for receptor-mediated transport of leptin into the brain. The purpose of this study was to determine whether ObRa is capable of transcellular transport of intact leptin. A transwell system in which Madin-Darby Canine Kidney (MDCK) cells stably expressing ObRa are grown in a monolayer was used to determine receptor distribution on apical or basolateral cell surfaces and the capacity for directional transport of 125I-leptin. Binding of 125I-leptin was greater on the apical vs. the basolateral cell surface and transport of 125I-leptin occurred only in the apical to basolateral direction. 11% of transported radioactivity appearing in the basolateral chamber represented intact leptin as assessed by TCA precipitation analysis and by SDS-PAGE. Parental MDCK cells did not express leptin receptors and did not bind or transport 125I-leptin. Epidermal growth factor (EGF) binding and transport via endogenous EGF receptors in MDCK cells also was assessed. In contrast to leptin, specific binding of 125I-EGF occurred primarily on the basolateral cell surface and transport of 125I-EGF occurred predominantly in the basolateral to apical direction. These data show that ObRa is preferentially targeted to the apical cell membrane in MDCK cells and that leptin transport occurs, albeit at a low rate, in a unidirectional manner in the apical to basolateral direction. These findings may be relevant to the putative role of ObRa in receptor-mediated transport of leptin from the circulation into the brain.