Neural stem cells persist in the adult mammalian brain in a neurogenic niche known as the subventricular zone (SVZ). SVZ neural stem cells (NSCs) can self-renew and are multipotent in culture. In rodents, adult NSCs correspond to SVZ astrocytes (type B cells) that are derived from radial glia, the NSCs of the embryonic and early postnatal brain. Type B cells generate transit-amplifying (type C) cells that give rise to young neurons (type A cells) and oligodendrocytes. Young neurons are born throughout the adult neurogenic niche and migrate tangentially through a complex network of chains that merge into the rostral migratory stream (RMS), a major pathway that leads into the olfactory bulb (OB). Within the OB, young neurons differentiate into multiple types of interneurons. The SVZ was thought to be limited to the lateral wall of the lateral ventricle, but recent work shows that the adult neurogenic niche is significantly more extensive and includes portions of the medial and dorsal walls of the lateral ventricle and the RMS itself. Furthermore, several recent studies explain why young OB neurons are generated in such an extensive region. Type B cells in different regions of the SVZ, although able to self-renew and generate both neurons and glial cells in vitro, are heterogeneous and committed to producing defined neuronal subtypes in vivo. The adult SVZ therefore provides a rich system to study not only neural replacement, but also the cellular and molecular mechanisms underlying regionalization and cell-fate specification.

Hematopoietic development is a complex process that involves a large number of growth factors and cytokines. Many cytokines are known to act on more mature, lineage-restricted cells of the hematopoietic system. However, no specific factors have yet been identified that induce the expansion of the most primitive hematopoietic cells without also inducing differentiation. To search for such factors, we isolated novel cell lines from the yolk sac in order to identify genes important in early hematopoietic and endothelial development. This approach led to the discovery of B219, a sequence that is expressed in at least four isoforms in very primitive hematopoietic cell populations and which may represent a novel hemopoietin receptor. The recently published receptor for the obesity (ob) gene product (leptin) is an isoform of B219 with a nearly identical ligand binding domain. B219/obr is expressed in the yolk sac, early fetal liver, enriched hematopoietic stem cells and in a variety of lymphohematopoietic cell lines. B219/obr is also expressed at high levels in adult reproductive organs. B219/obr maps to human chromosome 1p32, a region syntenic with the recently reported location of obr on murine chromosome 4 (ref. 5).

BACKGROUND

Leptin inhibits food intake and increases energy expenditure. Although the kidney expresses abundant transcripts of the short form of the leptin receptor (Ob-Ra), a role for this hormone in renal function remains unclear. Because individuals with massive obesity who may exhibit increased leptin serum concentrations develop renal glomerulosclerosis, we studied whether leptin can influence renal growth and profibrogenic processes.

METHODS

The effects of recombinant leptin on proliferation and synthesis of transforming growth factor-beta1 (TGF-beta1) was investigated in cultured glomerular endothelial cells of the rat (GERs) and syngeneic mesangial cells. Furthermore, leptin receptor expression and potential signal transduction pathways were evaluated in GERs. In addition, leptin was also infused for different time periods (72 hr and 3 weeks) into naive rats.

RESULTS

Recombinant mouse leptin induced proliferation of GERs, but not of syngeneic mesangial cells. Coincubation with angiotensin II and leptin exerts additive proliferative effects in GERs. An antileptin-receptor antibody totally abolished this proliferation but did not influence serum-induced proliferation. GER expressed high affinity receptors of the Ob-Ra type (Kd, 4 nM; Bmax, 9700 receptors/cell). Leptin also stimulated phosphorylation of STAT1alpha, and kinase inhibitors attenuated proliferation, suggesting a pivotal role of phosphorylation in this process. Incubation of GERs with leptin also induced mRNA expression of TGF-beta1 and enhanced secretion of this profibrogenic cytokine. Short-term leptin infusion (72 hr) into naive rats induced a significant proliferation, mainly restricted to glomerular endothelial cells, and enhanced glomerular TGF-beta1 mRNA levels. In rats continuously infused for three weeks with leptin, glomerular TGF-beta1 expression was still enhanced, and an additional increase in glomerular collagen type IV mRNA and protein expression was detected. These animals revealed an increase in proteinuria compared with control-infused rats.

CONCLUSIONS

Our findings are the first in vitro and in vivo demonstration that leptin is a renal growth and profibrogenic factor. These results may be an important contribution to our understanding of how leptin can contribute to renal damage, characterized by endocapillary proliferation and subsequent development of glomerulosclerosis, in pathophysiological situations with high circulating levels such as in diabetics or obese individuals. Although the effects of leptin itself are moderate, growth-promoting and profibrogenic effects may be enhanced in concert with other factors such as angiotensin II.

The biological role of macrophage infiltration into adipose tissue in obesity remains to be fully understood. We hypothesize that macrophages may act to stimulate angiogenesis in the adipose tissue. This possibility was examined by determining macrophage expression of angiogenic factor PDGF (platelet-derived growth factor) and regulation of tube formation of endothelial cells by PDGF. The data suggest that endothelial cell density was reduced in the adipose tissue of ob/ob mice. Expression of endothelial marker CD31 was decreased in protein and mRNA. The reduction was associated with an increase in macrophage infiltration. In the obese mice, PDGF concentration was elevated in the plasma, and its mRNA expression was increased in adipose tissue. Macrophages were found to be a major source of PDGF in adipose tissue, as deletion of macrophages led to a significant reduction in PDGF mRNA. In cell culture, PDGF expression was induced by hypoxia, and tube formation of endothelial cells was induced by PDGF. The PDGF activity was dependent on S6K, as inhibition of S6K in endothelial cells led to inhibition of the PDGF activity. We conclude that, in response to the reduced vascular density, macrophages may express PDGF in adipose tissue to facilitate capillary formation in obesity. Although the PDGF level is elevated in adipose tissue, its activity in angiogenesis is dependent on the availability of sufficient endothelial cells. The study suggests a new function of macrophages in the adipose tissue in obesity.

The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (k(obs)) was five to six orders of magnitude lower, whereas the affinity for the substrate (appK(d)) was reduced approximately 20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P.

The subventricular zone produces neuroblasts that migrate to the olfactory bulb (OB) and differentiate into interneurons throughout postnatal life (Altman and Das, 1966; Hinds, 1968; Altman, 1969; Kishi et al., 1990; Luskin, 1993; Lois and Alvarez-Buylla, 1994). Although such postnatally generated interneurons have been characterized morphologically, their physiological differentiation has not been thoroughly described. Combining retroviral-mediated labeling of newly generated neurons with patch-clamp electrophysiology, we demonstrated that soon after new cells enter the layers of the olfactory bulb, they display voltage-dependent currents typical of more mature neurons. We also show that these "newcomers" express functional GABA and glutamate receptor channels, respond synaptically to stimulation of the olfactory nerve, and may establish both axodendritic and dendrodendritic synaptic contacts within the olfactory bulb. These data provide a basic description of the physiology of newly generated cells in the OB and show that such new cells are functional neurons that synaptically integrate into olfactory bulb circuitry soon after their arrival.

Obesity presents a significant challenge to the general health of affluent nations in terms of the number of people affected, the serious associated maladies and the lack of effective treatments. While common wisdom has held that obesity results from 'gluttony and sloth', a number of studies have indicated physiological causes of underlying the pathogenesis of obesity, with the degree of adiposity having a strong genetic component. Recently, the obese gene in the ob/ob mouse was cloned, along with its human homologue. The specific production of the obese protein by adipose tissue suggested that it may function in a feedback loop from fat tissue to the hypothalamus to control energy intake and/or energy expenditure, and that it may play a role in the pathogenesis of human obesity. In this study we report that obese mRNA expression is elevated in ex vivo omental adipocytes isolated from massively obese humans in the absence of an identifiable mutation. Therefore, we speculate that this increased expression may suggest that the massively obese are insensitive to the putative regulatory function(s) of the obese gene product.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

The decline of leptin (Ob protein) concentrations during fasting is implicated as a signal for increasing the expression of the orexigenic peptide neuropeptide Y (NPY) in the hypothalamus. To test the hypothesis that the effects of food intake on arcuate nucleus NPY activation are mediated by leptin, we performed simultaneous triple in situ hybridization colocalization studies to determine whether the subset of NPY neurons that are activated by fasting preferentially expresses the long form of the leptin receptor (Ob-Rb). Thus, mRNAs encoding NPY and pro-opiomelanocortin (POMC) were colocalized in the arcuate nucleus of fed and fasted rats by fluorescence in situ hybridization in combination with isotopic in situ hybridization for Ob-Rb mRNA. In fed animals, 47% of arcuate nucleus neurons containing NPY mRNA also contained Ob-Rb mRNA, compared with 79% of POMC neurons (P < 0.01). After a 2-day fast, the number of arcuate nucleus neurons with NPY mRNA increased 50% (P < 0.05); the number of these that coexpressed Ob-Rb increased twofold (P = 0.013). Furthermore, Ob-Rb mRNA hybridization in individual NPY neurons increased by 64% (P < 0.02). In contrast, the number of POMC neurons that coexpressed Ob-Rb was unchanged. A significant interpretation of these findings is that the NPY neurons that do not express detectable levels of Ob-Rb mRNA are not activated by fasting, whereas the NPY neurons that are activated by fasting are the ones that express Ob-Rb. These data demonstrate a significant physiological difference between NPY neurons that express Ob-Rb and those that do not. The results support the conclusion that the effect of food intake on NPY neurons is mediated by the direct action of leptin via Ob-Rb receptors expressed by these NPY cells. The results also indicate that expression of Ob-Rb is a defining phenotypic characteristic of the subset of arcuate nucleus NPY neurons that are activated by fasting and play a central role in the adaptive response to negative energy balance.

Synchronized neural activity is believed to be essential for many CNS functions, including neuronal development, sensory perception, and memory formation. In several brain areas GABA(A) receptor-mediated synaptic inhibition is thought to be important for the generation of synchronous network activity. We have used GABA(A) receptor beta3 subunit deficient mice (beta3-/-) to study the role of GABAergic inhibition in the generation of network oscillations in the olfactory bulb (OB) and to reveal the role of such oscillations in olfaction. The expression of functional GABA(A) receptors was drastically reduced (>93%) in beta3-/- granule cells, the local inhibitory interneurons of the OB. This was revealed by a large reduction of muscimol-evoked whole-cell current and the total current mediated by spontaneous, miniature inhibitory postsynaptic currents (mIPSCs). In beta3-/- mitral/tufted cells (principal cells), there was a two-fold increase in mIPSC amplitudes without any significant change in their kinetics or frequency. In parallel with the altered inhibition, there was a significant increase in the amplitude of theta (80% increase) and gamma (178% increase) frequency oscillations in beta3-/- OBs recorded in vivo from freely moving mice. In odor discrimination tests, we found beta3-/- mice to be initially the same as, but better with experience than beta3+/+ mice in distinguishing closely related monomolecular alcohols. However, beta3-/- mice were initially better and then worse with practice than control mice in distinguishing closely related mixtures of alcohols. Our results indicate that the disruption of GABA(A) receptor-mediated synaptic inhibition of GABAergic interneurons and the augmentation of IPSCs in principal cells result in increased network oscillations in the OB with complex effects on olfactory discrimination, which can be explained by an increase in the size or effective power of oscillating neural cell assemblies among the mitral cells of beta3-/- mice.

Insulin resistance is a feature of many common disorders including obesity and type 2 diabetes mellitus. In these disorders, the beta-cells compensate for the insulin resistance for long periods of time with an increase in secretory capacity, an increase in beta-cell mass, or both. To determine whether the beta-cell response might relate to a circulating growth factor, we have transplanted normal islets under the kidney capsule of normoglycemic insulin-resistant mice with two different models of insulin resistance: lean mice that have a double heterozygous deletion of the insulin receptor and insulin receptor substrate-1 (DH) or the obese, hyperglycemic ob/ob mice. In the grafts transplanted into both hosts, there was a marked increase in beta-cell mitotic activity and islet mass that was comparable with that observed in the endogenous pancreas. By contrast, islets of the DH mouse transplanted into normal mice showed reduced mitotic index. These data suggest the insulin resistance is associated with a circulating islet cell growth factor that is independent of glucose and obesity.

Transgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was localized to the proximal tubule epithelium, and was associated with tubulointerstitial fibrosis, characterized by excessive collagen deposition and increased fibronectin and plasminogen activator inhibitor-1 immunoreactivity. Pronounced glomerulosclerosis was evident, and hydronephrosis developed with low penetrance. Expression of TGF-beta1 in white and brown adipose tissue resulted in a lipodystrophy-like syndrome. All white fat depots and brown fat pads were severely reduced in size, and exhibited prominent fibroplasia. This reduction in WAT was due to impaired adipose accretion. Introduction of the transgene into the ob/ob background suppressed the obesity characteristic of this mutation; however, transgenic mutant mice developed severe hepato- and splenomegaly. These studies strengthen the link between TGF-beta1 expression and fibrotic disease, and demonstrate the potency of TGF-beta1 in modulating mesenchymal cell differentiation in vivo.

Synchronized firing of mitral cells (MCs) in the olfactory bulb (OB) has been hypothesized to help bind information together in olfactory cortex (OC). In this survey of synchronized firing by suspected MCs in awake, behaving vertebrates, we find the surprising result that synchronized firing conveys information on odor value ("Is it rewarded?") rather than odor identity ("What is the odor?"). We observed that as mice learned to discriminate between odors, synchronous firing responses to the rewarded and unrewarded odors became divergent. Furthermore, adrenergic blockage decreases the magnitude of odor divergence of synchronous trains, suggesting that MCs contribute to decision-making through adrenergic-modulated synchronized firing. Thus, in the olfactory system information on stimulus reward is found in MCs one synapse away from the sensory neuron.

BACKGROUND

Tumor necrosis factor-alpha (TNF-alpha) is chronically elevated in the adipose tissue from obese humans and mice. This increase in TNF-alpha contributes to the insulin resistance, elevated plasminogen activator inhibitor-1 (PAI-1) levels, and cardiovascular complications associated with obesity and noninsulin-dependent diabetes (NIDDM). PAI-1 gene expression in adipose tissue is also stimulated by transforming growth factor-beta (TGF-beta). Experiments were performed to determine whether TGF-beta is regulated by TNF-alpha and elevated in obesity.

METHODS

The concentration of TGF-beta and PAI-1 mRNA in murine adipose tissue and cultured 3T3-L1 adipocytes was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), and the cellular localization of these molecules was evaluated using in situ hybridization and cell fractionation. Total TGF-beta protein was determined by employing an ELISA assay.

RESULTS

TGF-beta mRNA and protein were increased in the adipose tissue from two different strains of genetically obese mice (i.e., ob/ob and db/db), compared with their lean counterparts. This increase in TGF-beta may result from TNF-alpha since TNF-alpha increased TGF-beta mRNA expression in the adipose tissue of lean mice and stimulated TGF-beta production by cultured adipocytes. Administration of TGF-beta increased PAI-1 antigen in the plasma and PAI-1 mRNA in the adipocytes of lean mice, and enhanced the rate of PAI-1 synthesis by adipocytes in vitro.

CONCLUSIONS

TNF-alpha contributes to the elevated TGF-beta expression demonstrated in the adipose tissue of obese mice. A potential role for TGF-beta in the increased PAI-1 and vascular pathologies associated with obesity/NIDDM is suggested.

A missense mutation in the ob gene causes leptin deficiency and morbid obesity. Leptin replacement to three adults with this mutation normalized body weight and eating behavior. Because the neural circuits mediating these changes were unknown, we paired functional magnetic resonance imaging (fMRI) with presentation of food cues to these subjects. During viewing of food-related stimuli, leptin replacement reduced brain activation in regions linked to hunger (insula, parietal and temporal cortex) while enhancing activation in regions linked to inhibition and satiety (prefrontal cortex). Leptin appears to modulate feeding behavior through these circuits, suggesting therapeutic targets for human obesity.

Leptin is a multifunctional cytokine and hormone that primarily acts in the hypothalamus and plays a key role in the regulation of food intake and energy expenditure. In addition, it has direct effects on many cell types on the periphery. Leptin acts through its receptor, the product of the db gene, which has six isoforms. Only one of them (OB-Rb) has full signalling capabilities and is able to activate the Jak/STAT pathway, the major pathway used by leptin to exert its effects. However, some signalling events can be initiated by the short isoforms. Besides Jak/STAT, other pathways, such as MAPK and the 5'-AMP-activated protein kinase (AMPK) pathway, are also involved in leptin signalling. Leptin also interacts with insulin signalling. In this paper, we give an overview of the signal transduction mechanisms that are related to the actions of leptin.

This experiment determined the amount of leptin required to correct different abnormalities in leptin-deficient ob/ob mice. Baseline food intakes and body weights of lean (+/?) and obese (ob/ob) C57B1/6J (ob) mice were recorded for 7 days. An Alzet miniosmotic pump was placed in the peritoneal cavity of each mouse and delivered 0, 1, 2, 5, 10, or 42 microg/day human leptin for 7 days. In ob/ob mice, 2 microg leptin/day reduced food intake and body weight, and increased hypothalamic and brain stem serotonin concentrations. All fat pads were reduced 35-40% by 10 microg leptin/day, and liver weight, lipid, and glycogen decreased. Serum insulin and glucose were reduced in all leptin-treated ob/ob mice, and levels were normalized by 10 microg/day leptin. Low rectal temperatures of ob/ob mice were corrected by 10 and 42 microg/day leptin. These doses also increased brown adipose tissue uncoupling protein expression. The only responses in lean mice were a transient reduction in food intake and weight loss with 10 or 42 microg/day leptin. This study shows enhanced leptin sensitivity in ob/ob mice and suggests that increased temperature and sympathetic activity are indirect responses to high concentrations of protein.

Olfactory sensory neurons converge onto glomeruli in the olfactory bulb (OB) to form modular information processing units. Similar input modules are organized in translaminar columns for other sensory modalities. It has been less clear in the OB whether the initial modular organization relates to a columnar structure in the deeper layers involved in local circuit processing. To probe synaptic connectivity in the OB, we injected a retrograde-specific strain of the pseudorabies virus into the rat OB and piriform cortex. The viral-staining patterns revealed a striking columnar organization that extended across all layers of the OB from the glomeruli to the deep granule cell layer. We hypothesize that the columns represent an extension of the glomerular unit. Specific patterning was observed, suggesting selective, rather than distance-dependent, center-surround connectivity. The results provide a previously undescribed basis for interpreting the synaptic connections between mitral and granule cells within the context of a columnar organization in the OB and have implications for olfactory coding and network organization.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

The dynamics of sensory input to the nervous system play a critical role in shaping higher-level processing. In the olfactory system, the dynamics of input from olfactory receptor neurons (ORNs) are poorly characterized and depend on multiple factors, including respiration-driven airflow through the nasal cavity, odorant sorption kinetics, receptor-ligand interactions between odorant and receptor, and the electrophysiological properties of ORNs. Here, we provide a detailed characterization of the temporal organization of ORN input to the mammalian olfactory bulb (OB) during natural respiration, using calcium imaging to monitor ORN input to the OB in awake, head-fixed rats expressing odor-guided behaviors. We report several key findings. First, across a population of homotypic ORNs, each inhalation of odorant evokes a burst of action potentials having a rise time of about 80 ms and a duration of about 100 ms. This rise time indicates a relatively slow, progressive increase in ORN activation as odorant flows through the nasal cavity. Second, the dynamics of ORN input differ among glomeruli and for different odorants and concentrations, but remain reliable across successive inhalations. Third, inhalation alone (in the absence of odorant) evokes ORN input to a significant fraction of OB glomeruli. Finally, high-frequency sniffing of odorant strongly reduces the temporal coupling between ORN inputs and the respiratory cycle. These results suggest that the dynamics of sensory input to the olfactory system may play a role in coding odor information and that, in the awake animal, strategies for processing odor information may change as a function of sampling behavior.

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.

A key feature in the molecular pathogenesis of liver fibrosis requires maintenance of the activated hepatic stellate cell (HSC) phenotype by both proliferation and inhibition of apoptosis. We provide evidence that leptin is a potent HSC mitogen and dramatically inhibits stellate cell apoptosis. Leptin proved to be as potent an HSC mitogen as platelet-derived growth factor (PDGF) as assessed by bromodeoxyuridine (BrdU) incorporation in isolated primary HSCs; data using fluorescent propidium iodide (PI) uptake revealed that leptin, like PDGF, increased HSC populations in the S- and G2/M-phases of the cell cycle. Leptin resulted in a robust increase in cyclin D1 expression. Using the chemical inhibitor of Janus kinase 2 (Jak2) activity, AG 490, and overexpression of the suppressor of cytokine signaling 3 (SOCS-3), we show that blockade of leptin receptor (Ob-Rb) phosphorylation blocks leptin-induced HSC proliferation. Leptin-associated phosphorylation of both extracellular regulated kinase (p44/p42, Erk) and Akt is also prohibited. Further, the PI-3 kinase inhibitor LY294002 and MAPK inhibitor PD98059 were found to significantly reduce leptin-induced HSC proliferation, thereby indicating that leptin induced HSC proliferation is Akt- and Erk-dependent. Akt was also protective against HSC apoptosis. Leptin abolished both cycloheximide-induced and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, demonstrated by reduced caspase-3 activity, HSC-TUNEL staining, and DNA fragmentation. We conclude that leptin acts as a direct hepatic stellate cell survival agonist. Importantly, we have demonstrated that leptin-induced HSC proliferation and survival by Ob-Rb phosphorylation are both Erk- and Akt-dependent.

OBJECTIVE

The circulating peptide leptin produced by fat cells acts on central receptors to control food intake and body weight homeostasis. Contrary to initial reports, leptin expression has also been detected in the human placenta, muscles, and recently, in rat gastric chief cells. Here we investigate the possible presence of leptin and leptin receptor in the human stomach.

METHODS

Leptin and leptin receptor expression were assessed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot analysis on biopsy samples from 24 normal individuals. Fourteen (10 healthy volunteers and four patients with non-ulcer dyspepsia and normal gastric mucosa histology) were analysed for gastric secretions. Plasma and fundic mucosa leptin content was determined by radioimmunoassay.

RESULTS

In fundic biopsies from normal individuals, immunoreactive leptin cells were found in the lower half of the fundic glands. mRNA encoding ob protein was detected in the corpus of the human stomach. The amount of fundic leptin was 10.4 (3.7) ng leptin/g mucosa, as determined by radioimmunoassay. Intravenous infusions of pentagastrin or secretin caused an increase in circulating leptin levels and leptin release into the gastric juice. The leptin receptor was present in the basolateral membranes of fundic and antral gastric cells. mRNA encoding Ob-RL was detected in both the corpus and antrum, consistent with a protein of approximately 120 kDa detected by immunoblotting.

CONCLUSIONS

These data provide the first evidence of the presence of leptin and leptin receptor proteins in the human stomach and suggest that gastric epithelial cells may be direct targets for leptin. Therefore, we conclude that leptin may have a physiological role in the human stomach, although much work is required to establish this.

Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.