A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.

Leptin, the product of the ob gene, is thought to play a key role in the regulation of body fat mass. Beyond this function, it appears to be an integral component of various hypothalamo-pituitary-endocrine feedback loops. Because childhood and puberty are periods of major metabolic and endocrine changes, leptin levels and various hormonal parameters were investigated in a large cohort of healthy children and adolescents (312 males, 401 females, age 5.8-19.9 yr). For this purpose, a specific and sensitive RIA was developed that allowed the accurate measurement of low leptin levels in young lean children. With this assay, leptin proved to be a comparatively stable protein under common conditions of blood sampling and storage. Leptin levels increased in girls with age (r = 0.47, P < 0.0001), but decreased in boys (r = -0.34, P < 0.0001). An analysis according to pubertal stage showed a steady increase in girls between 2.51 micrograms/L (median) at Tanner stage 1 to 6.24 micrograms/L at Tanner stage 5. In boys, leptin levels were highest at Tanner stage 2 (2.19 micrograms/L) and declined thereafter to 0.71 microgram/L at Tanner stage 5. A strong exponential relationship was observed for leptin levels with body mass index (BMI) and percentage body fat as determined by bioelectric impedance measurements in a subgroup of subjects. This relationship was similar between boys and girls at Tanner stages 1 and 2. In boys, there was a significant decline of leptin at a given BMI with further progression of puberty that was much less pronounced in girls. Although the relative increase of leptin with BMI and percent body fat was the same in both genders, the absolute values at a given BMI or percent body fat were significantly lower in boys in late puberty and in adolescents. In boys, but not in girls, there was an inverse correlation with testosterone concentrations (r = -0.43, P < 0.0001), which explained 10.5% of the variation of leptin levels in a multiple regression model. Since BMI proved to be the major influencing variable, reference ranges were constructed using a best-fit regression line of the form leptin = a*e(b*BMI) and stratifying ranges according to gender and pubertal stage. In conclusion, these data suggest that 1) plasma leptin levels increase in girls and decrease in boys after Tanner stage 2 as the pubertal development proceeds; 2) they show a significant gender difference especially in late puberty and adolescence, even after adjustment for BMI or percent body fat; 3) the lower levels in males may be explained at least in part by a suppressive effect of androgens; 4) reference ranges with BMI as the independent variable should be stratified according to gender and pubertal stage.

The OB-fold is found in all three kingdoms and is well represented in both sequence and structural databases. The OB-fold is a five-stranded closed beta barrel and the majority of OB-fold proteins use the same face for ligand binding or as an active site. Different OB-fold proteins use this 'fold-related binding face' to, variously, bind oligosaccharides, oligonucleotides, proteins, metal ions and catalytic substrates. Recently, a number of new structures with OB-folds have been reported that augment the variation seen for this set of proteins whilst conserving the characteristic fold and binding face. The conservation of fold and a functional binding face amongst many structures provides a model for investigating the evolutionary trajectory of sequence, structure and function.

Our understanding of the cellular and molecular mechanisms underlying the adult neural stem cell state remains fragmentary. To provide new models on this issue, we searched for stem cells in the adult brain of the zebrafish. Using BrdU tracing and immunodetection of cell-type-specific markers, we demonstrate that the adult zebrafish telencephalon contains self-renewing progenitors, which show features of adult mammalian neural stem cells but distribute along the entire dorso-ventral extent of the telencephalic ventricular zone. These progenitors give rise to newborn neurons settling close to the ventricular zone within the telencephalon proper. They have no equivalent in mammals and therefore constitute a new model of adult telencephalic neural stem cells. In addition, progenitors from the ventral subpallium generate rapidly dividing progenitors and neuroblasts that reach the olfactory bulb (OB) via a rostral migratory stream and differentiate into GABAergic and TH-positive neurons. These ventral progenitors are comparable to the mammalian neural stem cells of the subependymal zone. Interestingly, dorsal and ventral progenitors in the adult telencephalon express a different combination of transcription factors than their embryonic counterparts. In the case of neurogenin1, this is due to the usage of different enhancer elements. Together, our results highlight the conserved and unique phylogenic and ontogenic features of adult neurogenesis in the zebrafish telencephalon and open the way to the identification of adult neural stem cell characters in cross-species comparative studies.

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased>> 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Leptin, the obese (ob) gene product, is thought to be a lipostatic hormone that contributes to body weight regulation through modulating feeding behavior and/or energy expenditure. The determinants of plasma leptin concentration were evaluated in 267 subjects (106 with normal glucose tolerance, 102 with impaired glucose tolerance, and 59 with noninsulin-dependent diabetes). Fasting plasma leptin levels ranged from 1.8-79.6 ng/mL (geometric mean, 12.4), were higher in the obese subjects, and were not related to glucose tolerance. Women had approximately 40% higher leptin levels than men at any level of adiposity. After controlling for body fat, postmenopausal women had still higher leptin levels than men of similar age, and their levels were not different from those in younger women. Multiple regression analysis showed that adiposity, gender, and insulinemia were significant determinants of leptin concentration, explaining 42%, 28%, and 2% of its variance, respectively. Neither age nor the waist/hip ratio was significantly related to leptin concentration. Thus, our data indicate that gender is a major determinant of the plasma leptin concentration. This sex difference is not apparently explained by sex hormones or body fat distribution. Leptin's sexual dimorphism suggests that women may be resistant to its putative lipostatic actions and that it may have a reproductive function.

The neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons of the hypothalamic arcuate nucleus are thought to promote feeding. Here, we demonstrate that feeding state in vivo, through a leptin-dependent process, induces large and persistent changes in the electrophysiological activity of these neurons as measured extracellularly in vitro. Consistent with an orexigenic role, fasting induced a 4-fold increase in the basal action potential frequency of NPY/AgRP neurons. Leptin, when injected into fasted wild-type mice, induced a dose- and time-dependent decrease in spike frequency, which approached fed levels 2-3 h post treatment. In leptin-deficient (lep(ob)/lep(ob)) and leptin receptor-deficient (lepr(db)/lepr(db)) mice, NPY/AgRP spike frequency was not significantly increased by fasting, and even in mutant mice fed ad libitum, spike frequency was at least as high as in fasted wild-type mice. All recordings included GABA(A) and ionotropic glutamate receptor antagonists, suggesting that expression of this modulation is potentially intrinsic and not synaptically dependent. Recorded neurons were unambiguously identified using NPY-Sapphire transgenic mice. This is a remarkably straightforward example of a very robust in vitro electrophysiogical effect produced by a simple behavioral manipulation, food restriction.

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results-obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers-indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.

We used a 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mouse to study postnatal subventricular zone (SVZ) progenitor fate, with a focus on the olfactory bulb (OB). The postnatal OB of the CNP-EGFP mouse contained EGFP+ interneurons and oligodendrocytes. In the anterior SVZ, the majority of EGFP+ progenitors were NG2+. These NG2+/EGFP+ progenitors expressed the OB interneuron marker Er81, the neuroblast markers doublecortin (DC) and Distalless-related homeobox (DLX), or the oligodendrocyte progenitor marker Nkx2.2. In the rostral migratory stream (RMS), EGFP+ cells displayed a migrating phenotype. A fraction of these cells were either NG2-/Er81+/DC+/DLX+ or NG2+/Nkx2.2+. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) injection into the lateral ventricle (LV) of early postnatal mice demonstrated that NG2+/EGFP+ progenitors migrate from the SVZ through the RMS into the OB. Moreover, fluorescence-activated cell-sorting-purified NG2+/CNP-EGFP+ or NG2+/beta-actin-enhanced yellow fluorescent protein-positive (EYFP+) progenitors transplanted into the early postnatal LV displayed extensive rostral and caudal migration. EYFP+ or EGFP+ graft-derived cells within the RMS were DLX+/Er81+ or Nkx2.2+, migrated to the OB, and differentiated to interneurons and oligodendrocytes. In the subcortical white matter (SCWM), grafted cells differentiated to either oligodendrocytes or astrocytes. Transplantation of NG2+/EYFP+ progenitors selectively purified from the SVZ showed that these cells were migratory and generated glia and neurons in the OB, hippocampus, and striatum. In contrast, cortical, OB, or cerebellar NG2+ cells had a very limited migratory potential and gave rise to glia in the SCWM and striatum. Our findings indicate region-specific differences between NG2+ progenitor cells and show that NG2+ cells can migrate throughout the RMS and contribute to both gliogenesis and neurogenesis in the postnatal OB.

Whereas functional, metabolic, neurotrophic, and morphological abnormalities of peripheral diabetic neuropathy (PDN) have been extensively explored in streptozotocin-induced diabetic rats and mice (models of type 1 diabetes), insufficient information is available on manifestations and pathogenetic mechanisms of PDN in type 2 diabetic models. The latter could constitute a problem for clinical trial design because the vast majority of subjects with diabetes have type 2 (non-insulin dependent) diabetes. This study was aimed at characterization of PDN in leptin-deficient (ob/ob) mice, a model of type 2 diabetes with relatively mild hyperglycemia and obesity. ob/ob mice ( approximately 11 weeks old) clearly developed manifest sciatic motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable ( approximately 78%) loss of intraepidermal nerve fibers. They also had increased sorbitol pathway activity in the sciatic nerve and increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion (DRG). Aldose reductase inhibition with fidarestat (16 mg . kg(-1) . d(-1)), administered to ob/ob mice for 6 weeks starting from 5 weeks of age, was associated with preservation of normal MNCV and SNCV and alleviation of thermal hypoalgesia and intraepidermal nerve fiber loss but not tactile allodynia. Sciatic nerve nitrotyrosine immunofluorescence and the number of poly(ADP-ribose)-positive nuclei in sciatic nerve, spinal cord, and DRGs of fidarestat-treated ob/ob mice did not differ from those in nondiabetic controls. In conclusion, the leptin-deficient ob/ob mouse is a new animal model that develops both large motor and sensory fiber and small sensory fiber PDN and responds to pathogenetic treatment. The results support the role for increased aldose reductase activity in functional and structural changes of PDN in type 2 diabetes.

Recent evidence indicates that leptin modifies T cell immunity, and may provide a key link between nutritional deficiency and immune dysfunction. To study the influence of leptin on autoimmunity, susceptibility to experimental autoimmune encephalomyelitis induced by immunization with a myelin-derived peptide was examined in leptin-deficient, C57BL/6J-ob/ob mice, with or without leptin replacement, and in wild-type controls. Leptin replacement converted disease resistance to susceptibility in the C57BL/6J-ob/ob mice; this was accompanied by a switch from a Th2 to Th1 pattern of cytokine release and consequent reversal of Ig subclass production. Our findings suggest that leptin is required for the induction and maintenance of an effective proinflammatory immune response in the CNS.

A main function of white adipose tissue is to release fatty acids from stored triacylglycerol for other tissues to use as an energy source. Whereas endocrine regulation of lipolysis has been extensively studied, autocrine and paracrine regulation is not well understood. Here we describe the role of the newly identified major adipocyte phospholipase A(2), AdPLA (encoded by Pla2g16, also called HREV107), in the regulation of lipolysis and adiposity. AdPLA-null mice have a markedly higher rate of lipolysis owing to increased cyclic AMP levels arising from the marked reduction in the amount of adipose prostaglandin E(2) that binds the Galpha(i)-coupled receptor, EP3. AdPLA-null mice have markedly reduced adipose tissue mass and triglyceride content but normal adipogenesis. They also have higher energy expenditure with increased fatty acid oxidation within adipocytes. AdPLA-deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and is crucial for the development of obesity.

Recognition of discrete commitment and differentiation stages requires characterization of changes in proliferative capacity together with the temporal acquisition or loss of expression of molecular and morphological traits. Both cell lines and primary cultures have been useful for analysis of transitional steps in the chondroblast (CB) and osteoblast (OB) lineages. One striking feature is that OBs and CBs share expression of some molecules, including newer markers such as epsilon BP (galectin-3), while also having unique markers. The fact that hypertrophic chondrocytes appear able to downregulate cartilage markers and upregulate OB markers also points to an interesting lineage relationship that needs to be explored further. Recently, we have focused on the osteoprogenitors that divide and differentiate into mature OBs forming bone nodules in fetal rat calvaria cell cultures. We use cellular, immunocytochemical, and molecular approaches, including PCR on small numbers of cells, to discriminate stages. Nodule formation is characterized by loss of proliferative capacity and sequential increased marker expression, that is, alkaline phosphatase (AP), followed by bone sialoprotein (BSP), and osteocalcin. Upregulation of collagen type I and biphasic expression of osteopontin, with two peaks corresponding to proliferation and differentiation stages, also occurs. A variety of other molecules are also upregulated in the mature OB, including epsilon BP and CD44s. By replica plating and PCR, we have begun to study the expression of the messenger RNAs (mRNAs) for potential regulatory molecules (e.g., PTHrP) and their receptors (e.g., PTHR, FGFR-1, and PDGFR alpha) and have found all to be modulated during the progression from committed osteoprogenitor to mature OB.(ABSTRACT TRUNCATED AT 250 WORDS)

Adiponutrin and a related protein, adipocyte triglyceride lipase (ATGL; also known as Desnutrin), were recently described as adipocyte-specific proteins with lipid hydrolase activity. Using bioinformatics, we identified three additional Adiponutrin family members (GS2, GS2-Like, and PNPLA1). Here, we report on the expression, regulation, and activity of GS2 and GS2-Like compared with Adiponutrin and Desnutrin/ATGL. GS2-Like is expressed and regulated in a manner similar to Adiponutrin; however, the absolute levels of mRNA are significantly lower than those of Adiponutrin or Desnutrin/ATGL. GS2 transcripts were identified only in humans and are highly expressed in adipose as well as other tissues. All four proteins show lipase activity in vitro, which is dependent on the presence of the active site serine for Adiponutrin, Desnutrin/ATGL, and GS2. Overexpression of Desnutrin/ATGL, GS2, and GS2-Like, but not Adiponutrin, decreases intracellular triglyceride levels. This is consistent with a function for Desnutrin/ATGL, GS2, and GS2-Like in lipolysis, but not for Adiponutrin. Consistent with previously reported data, Desnutrin/ATGL is upregulated by fasting in adipose tissue, whereas Adiponutrin is downregulated. Additionally, Adiponutrin and GS2-Like, but not Desnutrin/ATGL, are strongly induced in the liver of ob/ob mice. Our data support distinct functions for Adiponutrin and Desnutrin/ATGL and raise the possibility that GS2 may contribute significantly to lipolysis in human adipose tissue.

Normal mice and leptin-deficient ob/ob mice were treated with leptin to study effects on osteogenesis and adipogenesis in bone marrow. Leptin treatment significantly decreased bone marrow adipocyte size and number in ob/ob mice while increasing bone formation, BMC, and BMD. The results suggest that, in leptin-sensitive animals, the reduction in marrow adipocytes has positive effects on bone formation.

BACKGROUND

Adipocytes, osteoblasts, and osteoclasts have leptin receptors, and leptin can also affect bone metabolism indirectly through its receptors in the hypothalamus. We examined the effects of leptin treatment on bone formation, BMD, and marrow adipocyte population in normal mice and leptin-deficient ob/ob mice.

METHODS

At the age of 15 weeks, mice were implanted with Alzet osmotic pumps for subcutaneous delivery of treatment solutions (saline, 2.5 microg leptin/day, or 10 microg leptin/day) for 14 days at a delivery rate of 0.25 microl/h. Bone formation was assessed using fluorochrome labels, cell populations were quantified using histomorphometry, and bone densitometry was measured using DXA. We also used a Luminex Beadlyte assay system to quantify cell survival markers in bone marrow samples.

CONCLUSIONS

Results indicate that both doses of leptin decreased the number of marrow adipocytes in ob/ob mice by >20% (p < 0.05) compared with PBS-treated ob/ob mice. The decrease in adipocyte number with leptin treatment is accompanied by an increase in concentration of the apoptosis marker caspase-3 in bone marrow adipocytes and hematopoietic cells. Both leptin doses also significantly (p < 0.05) increased the percentage of fluorochrome-labeled tibial endosteal surface by >30% compared with PBS-treated ob/ob mice. Leptin treatment increased whole body BMC by >30% in the ob/ob mice receiving the highest leptin dose. Leptin treatment provided no increase in bone formation, BMC, or BMD in normal, leptin-replete mice.

Obesity is accompanied by complications such as hypertension, non-insulin-dependent diabetes mellitus and atherosclerosis, which in turn cause ischaemic heart disease, stroke and premature death. The underlying mechanisms behind imbalance in energy intake and energy expenditure that lead to obesity are still controversial. In most populations, obesity is more common among women than men and is a multifactorial phenotype, which may result from a complex network of genetic and nongenetic factors. The relative importance of genetic factors for obesity is under debate. Genome searches using polymorphic markers in inbred mice with phenotypes that result in extreme obesity and studies of human candidate genes are being performed in an attempt to identify genes that contribute to obesity. There is evidence that body weight is physiologically regulated and it has been postulated that the storage of fat may provide signals to the brain that the body is obese, which in turn may make the subject eat less and burn more fuel. One of the molecules that may be involved in such signalling is the obese (ob) gene product. Mutations in ob result in profound obesity and type II diabetes in mice. The mouse ob gene and its human homologue have been cloned and sequenced. The gene is expressed in adipose tissue and the product has features of a secreted protein. We have investigated human ob expression in subcutaneous and omental adipose tissue obtained from non-obese and massively obese subjects using in situ hybridization histochemistry and report on overexpression in obese people.

Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding, and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here, we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13 A pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.

OBJECTIVE

Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), is an appetite stimulatory signal from the stomach with structural resemblance to motilin. We examined the effects of the gastric peptide ghrelin and GHS-R antagonists on energy balance and glycaemic control in mice.

METHODS

Body weight, fat mass, glucose, insulin, and gene expression of leptin, adiponectin, and resistin in white adipose tissue (WAT) were measured after repeated administrations of ghrelin under a high fat diet. Gastric ghrelin gene expression was assessed by northern blot analysis. Energy intake and gastric emptying were measured after administration of GHS-R antagonists. Repeated administration of GHS-R antagonist was continued for six days in ob/ob obese mice.

RESULTS

Ghrelin induced remarkable adiposity and worsened glycaemic control under a high fat diet. Pair feeding inhibited this effect. Ghrelin elevated leptin mRNA expression and reduced resistin mRNA expression. Gastric ghrelin mRNA expression during fasting was increased by a high fat diet. GHS-R antagonists decreased energy intake in lean mice, in mice with diet induced obesity, and in ob/ob obese mice; it also reduced the rate of gastric emptying. Repeated administration of GHS-R antagonist decreased body weight gain and improved glycaemic control in ob/ob obese mice.

CONCLUSIONS

Ghrelin appears to be closely related to excess weight gain, adiposity, and insulin resistance, particularly under a high fat diet and in the dynamic stage. Gastric peptide ghrelin and GHS-R may be promising therapeutic targets not only for anorexia-cachexia but also for obesity and type 2 diabetes, which are becoming increasingly prevalent worldwide.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

With the growing prevalence of obesity, scientific interest in the biology of adipose tissue has been extended to the secretory products of adipocytes, since they are increasingly shown to affect several aspects in the pathogenesis of obesity-related diseases. The cloning of the ob gene is consistent with this concept and suggests that body fat content in adult rodents is regulated by a negative feedback loop centred in the hypothalamus. In recent years, a number of additional signalling molecules secreted by adipose tissue have been discovered, commonly referred to as 'adipocytokines'. Among these, adiponectin is perhaps the most interesting and promising compound for the clinician since it has profound protective actions in the pathogenesis of diabetes and cardiovascular disease. Adiponectin is low in obese subjects and, in particular, insulin-resistant patients. In contrast, resistin seems to be of greater relevance in relation to the immune stress response than in the regulation of glucose homeostasis. However, inflammatory processes have recently been connected with the development of atherosclerosis. Finally, little is known regarding the clinical relevance of visfatin. Recent research has revealed many functions of adipocytokines extending far beyond metabolism, such as immunity, cancer and bone formation. This report aims to review some of the recent topics of adipocytokine research that may be of particular importance.

To identify new genes that are important in fat metabolism, we utilized the Lexicon-Genentech knockout database of genes encoding transmembrane and secreted factors and whole murine genome transcriptional profiling data that we generated for 3T3-L1 in vitro adipogenesis. Cross-referencing null models evidencing metabolic phenotypes with genes induced in adipogenesis led to identification of a new gene, which we named RIFL (refeeding induced fat and liver). RIFL-null mice have serum triglyceride levels approximately one-third of wild type. RIFL transcript is induced >100-fold during 3T3-L1 adipogenesis and is also increased markedly during adipogenesis of murine and human primary preadipocytes. siRNA-mediated knockdown of RIFL during 3T3-L1 adipogenesis results in an ~35% decrease in adipocyte triglyceride content. Murine RIFL transcript is highly enriched in white and brown adipose tissue and liver. Fractionation of WAT reveals that RIFL transcript is exclusive to adipocytes with a lack of expression in stromal-vascular cells. Nutritional and hormonal studies are consistent with a prolipogenic function for RIFL. There is evidence of an approximately eightfold increase in RIFL transcript level in WAT in ob/ob mice compared with wild-type mice. RIFL transcript level in WAT and liver is increased ~80- and 12-fold, respectively, following refeeding of fasted mice. Treatment of 3T3-L1 adipocytes with insulin increases RIFL transcript ≤35-fold, whereas agents that stimulate lipolysis downregulate RIFL. Interestingly, the 198-amino acid RIFL protein is predicted to be secreted and shows ~30% overall conservation with the NH(2)-terminal half of angiopoietin-like 3, a liver-secreted protein that impacts lipid metabolism. In summary, our data suggest that RIFL is an important new regulator of lipid metabolism.

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.