No abstract

We have recently described a molecular gatekeeper of the hypothalamic-pituitary-gonadal axis with the observation that G protein-coupled receptor 54 (GPR54) is required in mice and men for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion to occur. In the present study, we investigate the possible central mode of action of GPR54 and kisspeptin ligand. First, we show that GPR54 transcripts are colocalized with gonadotropin-releasing hormone (GnRH) neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, may act directly on these neurons. Next, we show that GnRH neurons seem anatomically normal in gpr54 ؊/؊ mice, and that they show projections to the median eminence, which demonstrates that the hypogonadism in gpr54 ؊/؊ mice is not due to an abnormal migration of GnRH neurons (as occurs with KAL1 mutations), but that it is more likely due to a lack of GnRH release or absence of GnRH neuron stimulation. We also show that levels of kisspeptin injected i.p., which stimulate robust LH and FSH release in wild-type mice, have no effect in gpr54 ؊/؊ mice, and therefore that kisspeptin acts directly and uniquely by means of GPR54 signaling for this function. Finally, we demonstrate by direct measurement, that the central administration of kisspeptin intracerebroventricularly in sheep produces a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin on the hypothalamo-pituitary-gonadal axis occurs directly at the level of GnRH release. The localization and GnRH release effects of kisspeptin thus define GPR54 as a major control point in the reproductive axis and suggest kisspeptin to be a neurohormonal effector.hypothalamus ͉ neurons ͉ gonadotropin axis ͉ reproduction

Mutations in GPR54, a G protein-coupled receptor gene, cause autosomal recessive idiopathic hypogonadotropic hypogonadism in humans and mice, suggesting that this receptor is essential for normal gonadotropin-releasing hormone physiology and for puberty.

stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model, Pomc ؊/؊ mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from Pomc ؊/؊ mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both Pomc ؊/؊ and Pomc ؉/؊ mice. The effects of leptin on food intake and body weight were blunted in obese Pomc ؊/؊ mice whereas nonobese Pomc ؊/؊ mice were sensitive to leptin. Surprisingly, we found that Pomc ؊/؊ mice maintained their acute anorectic response to peptide-YY3-36 (PYY3-36). However, 7 days of PYY3-36 administration had no effect on cumulative food intake or body weight in wild-type or Pomc ؊/؊ mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY3-36 or full effects of leptin on feeding. The finding that the loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity. H ypothalamic neurons expressing pro-opiomelanocortin (POMC) are involved in the maintenance of energy homeostasis through the integration of a number of peripheral and central signals related to energy status (1-3). POMC is highly expressed in neuronal cell bodies of the arcuate nucleus, with POMC-expressing neurons innervating other hypothalamic regions known to regulate feeding behavior, including the paraventricular nucleus, lateral hypothalamus, and dorsomedial hypothalamic nucleus (DMH) (4). Pharmacological and genetic studies have revealed that POMCderived melanocortin peptides or synthetic agonists suppress feeding through activation of the melanocortin 4-receptor (MC4R) whereas the endogenous antagonist agouti-related protein (AgRP) or synthetic antagonists stimulate food intake (5).Approximately 40% of POMC-expressing neurons colocalize with the long isoform of the leptin receptor (6). Leptin activates POMC neurons directly and indirectly, through hyperpolarization of neuropeptide Y (NPY)͞AgRP neurons, thereby reducing their tonic inhibitory ␥-aminobutyric acid (GABA) input to POMC neurons (1). Recent data suggest that the gut peptide PYY 3-36 (peptide-YY 3-36 ) reduces food intake, at least in part, through direct hyperpolarization of NPY͞AgRP neurons and subsequent disinhibition of POMC neurons (3).Given the critical role that hypothalamic POMC plays in the regulation of energy balance, it is not surprising that m...

The nuclear receptor peroxisome proliferator-activated receptor-␥ (PPAR␥) is critically required for adipogenesis. PPAR␥ exists as two isoforms, ␥1 and ␥2. PPAR␥2 is the more potent adipogenic isoform in vitro and is normally restricted to adipose tissues, where it is regulated more by nutritional state than PPAR␥1. To elucidate the relevance of the PPAR␥2 in vivo, we generated a mouse model in which the PPAR␥2 isoform was specifically disrupted. Despite similar weight, body composition, food intake, energy expenditure, and adipose tissue morphology, male mice lacking the ␥2 isoform were more insulin resistant than wild-type animals when fed a regular diet. These results indicate that insulin resistance associated with ablation of PPAR␥2 is not the result of lipodystrophy and suggests a specific role for PPAR␥2 in maintaining insulin sensitivity independently of its effects on adipogenesis. Furthermore, PPAR␥2 knockout mice fed a high-fat diet did not become more insulin resistant than those on a normal diet, despite a marked increase in their mean adipocyte cell size. These findings suggest that PPAR␥2 is required for the maintenance of normal insulin sensitivity in mice but also raises the intriguing notion that PPAR␥2 may be necessary for the adverse effects of a high-fat diet on carbohydrate metabolism. Diabetes 54: 1706 -1716, 2005 P eroxisome proliferator-activated receptor-␥ (PPAR␥) plays a central role in adipogenesis and insulin sensitivity. PPAR␥ is expressed as two isoforms, PPAR␥1 and PPAR␥2, which differ only in that PPAR␥2 has 30 extra amino acids at its NH 2 terminus. Under physiological conditions, PPAR␥2 is expressed almost exclusively in white and brown adipocytes, whereas PPAR␥1 is also expressed in colon, macrophages, skeletal muscle, and liver (1). Although there is limited information regarding the functional differences between these two splice variants, PPAR␥2 may be more adipogenic than PPAR␥1 (2,3). PPAR␥2 may play a distinct role in regulating insulin sensitivity as suggested by the strong epidemiological evidence that the PPAR␥2-specific Pro12Ala variant influences diabetes susceptibility in humans (4).We have shown that expression of PPAR␥ isoforms is differentially regulated by nutritional factors (5). Murine studies showed that PPAR␥2 mRNA is markedly downregulated in white adipose tissue (WAT) by fasting and normalized by re-feeding (1). Similarly, PPAR␥2 gene expression is increased in WAT by a high-fat diet (HFD) as well as in mouse models of diet-induced obesity (5). Studies using genetically modified mouse models have addressed the role of PPAR␥ in vivo (6). A proadipogenic role for PPAR␥ in vivo was supported by the global PPAR␥-deficient and the hypomorphic PPAR␥ mouse models (7-9). In addition to a role in promoting adipogenesis, activation of PPAR␥ also improves insulin sensitivity (10). However, the characterization of the heterozygous PPAR␥ knockout mouse provided the paradoxical finding that mice with a 50% reduction in PPAR␥ gene dosage were resistant to HFD-induced ob...

Peg3 encodes a C2H2 type zinc finger protein that is implicated in a novel physiological pathway regulating core body temperature, feeding behavior, and obesity in mice. Peg3+/- mutant mice develop an excess of abdominal, subcutaneous, and intra-scapular fat, despite a lifetime of lower food intake than wild-type animals. However, they start life with reduced fat reserves and are slower to enter puberty. These mice maintain a lower core body temperature, fail to respond to a cold challenge, and have lower metabolic activity as measured by oxygen consumption. Plasma leptin levels are significantly higher than in wild types, and Peg3+/- mice appear to have developed leptin resistance. Administration of exogenous leptin resulted in a significant reduction in food intake in wild-type mice that was not observed in Peg3+/- mutants. This mutation, which is strongly expressed in hypothalamic tissue during development, has the capacity to regulate multiple events relating to energy homeostasis.

MLL5 is a divergent member of the Drosophila Trithorax-related (SET) domain and plant homeodomain (PHD) domaincontaining chromatin regulators that are involved in the regulation of transcriptional "memory" during differentiation. Human MLL5 is located on chromosome 7q22, which frequently is deleted in myeloid leukemias, suggesting a possible role in hemopoiesis. To address this question, we generated a loss-of-function allele (Mll5 tm1Apa ) in the murine Mll5 locus. Unlike other Mll genes, Mll5 tm1Apa homozygous mice are viable but display defects in immunity and hematopoiesis. First, Mll5 tm1Apa homozygous mice show increased susceptibility to spontaneous eye infections, associated with a cellautonomous impairment of neutrophil function. Second, Mll5 tm1Apa/tm1Apa mice exhibit a mild impairment of erythropoiesis. Third, Mll5 tm1Apa/tm1Apa hematopoietic stem cells (HSCs) have impaired competitive repopulating capacity both under normal conditions and when subjected to selfrenewal stimulation by NUP98-HOXA10. Fourth, Mll5 tm1Apa homozygous HSCs show a dramatic sensitivity to DNA demethylation-induced differentiation (5-azadeoxycytidine). Taken together, our data show that MLL5 is involved in terminal myeloid differentiation and the regulation of HSC self-renewal by a mechanism that involves DNA methylation. These data warrant investigation of MLL5 expression levels as a predictive marker of demethylating-agent response in patients with myelodysplastic syndromes and leukemias and identify MLL5 as a key regulator of normal hematopoiesis. IntroductionThe mammalian MLL protein (from original identification in mixed lineage leukemias, also known as TRX or ALL1 and now classified as MLL1) is the just identified member of the MLL family comprising 5 members (MLL1-5) that are thought to regulate stable states of transcription during developmental processes. MLL1-4 proteins share the greatest similarity with the Drosophila Trithorax group, 1 but all 5 members contain at least 1 conserved Su(var)3,9, enhancer of zest, Trithorax (SET) domain, and plant homeodomain (PHD) zinc finger motif. SET domains possess histone methyltransferase activity, 2,3 whereas PHD fingers are binding/recognition motifs for histone modifications. [4][5][6] The SET domain of MLL1 was found to have H3K4-specific histone methyltransferase activity 7,8 and, like MLL2 (now classified as MLL4(TRX2)), was found in chromatin remodeling complexes containing menin. 9 MLL3 and MLL4 (now classified as MLL3(HALR) and MLL2(ALR), respectively) are found in complexes containing ASC-2 10 and are associated with H3 Lys-27 (K27)-specific demethylating activity by UTX. 11 MLL (MLL1) also can directly and indirectly regulate DNA methylation. 12 MLL5 initially was assigned to this family based on the sequence homology of the SET domain; however, the overall sequence similarity suggests a closer relationship to yeast SET3 and SET4 proteins. 13,14 Currently, no experimental evidence exists of SET methyltransferase activity associated with any member of the yeast SET3/...