Alterations in the production of or the sensitivity to leptin, the protein encoded by the ob gene, cause obesity and diabetes in rodents. We evaluated the isolated relationship between leptin and insulin sensitivity in lean and obese humans. Three groups of subjects who were carefully matched for either insulin sensitivity (determined by the modified intravenous glucose tolerance test and minimal model analysis) or adiposity (determined by hydrodensitometry) were studied: 1) lean insulin-sensitive men (percentage body fat, 15 +/- 1%); 2) lean insulin-resistant men (percentage body fat, 16 +/- 1%), matched on percentage body fat and fat mass with the lean insulin-sensitive group; and 3) obese insulin-resistant men (percentage body fat, 31 +/- 3), matched on insulin sensitivity with the lean insulin-resistant group. Basal plasma leptin concentrations were significantly lower in the lean insulin-sensitive than in the lean insulin-resistant men (1.90 +/- 0.4 vs. 4.35 +/- 1.21 ng/ml, P < 0.05) despite identical body composition. Plasma leptin in the obese men (9.27 +/- 1.4 ng/ml) was significantly higher than values in the two lean groups (P < 0.01). Marked alterations in plasma glucose and insulin concentrations induced by glucose and tolbutamide injection did not cause any change in plasma leptin levels. These results demonstrate that insulin resistance is associated with elevated plasma leptin levels independent of body fat mass. However, plasma insulin itself does not acutely regulate leptin production.

PNPLA3 (adiponutrin), a novel patatin-like phospholipase domain-containing enzyme, is expressed at high level in fat, but also in other tissues including liver. Polymorphisms in PNPLA3 have been linked to obesity and insulin sensitivity. Notably, a nonsynonymous variant rs738409(G) allele of the PNPLA3 gene was found to be strongly associated with both nonalcoholic and alcoholic fatty liver disease. We have generated Pnpla3(-/-) mice by gene targeting. Loss of Pnpla3 has no effect on body weight or composition, adipose mass, or development, whether the mice were fed regular chow or high-fat diet or bred into the genetic obese Lep(ob/ob) background. Plasma and liver triglyceride content and plasma aspartate aminotransferase and alanine aminotransferase levels were not different between Pnpla3(+/+) and Pnpla3(-/-) mice while they were on regular chow, fed three different fatty liver-inducing diets, or after they were bred into Lep(ob/ob) background. Hepatic Pnpla5 messenger RNA (mRNA) levels were similar in wild-type and Pnpla3(-/-) mice, although adipose Pnpla5 mRNA level was increased in Pnpla3(-/-) mice. A high-sucrose lipogenic diet stimulated hepatic Pnpla3 and Pnpla5 mRNA levels to a similar degree, but it did not affect adipose or liver triglyceride lipase (ATGL, known also as Pnpla2) mRNA in Pnpla3(+/+) and Pnpla3(-/-) mice. Finally, Pnpla3(+/+) and Pnpla3(-/-) mice displayed similar glucose tolerance and insulin tolerance tests while on regular chow or three different fatty liver-inducing diets.

CONCLUSIONS

Loss of Pnpla3 does not cause fatty liver, liver enzyme elevation, or insulin resistance in mice.

Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout an individual's lifetime. Interaction of HSCs with their specific microenvironments, known as stem cell niches, is critical for maintaining stem cell properties, including self-renewal capacity and the ability to differentiate into multiple lineages. During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of HSCs in specialized microenvironmental niches. In the adult BM, HSCs are located in the trabecular endosteum (osteoblastic niche) or sinusoidal perivascular (vascular niche) areas. Here we show that osteoblastic cells (OBs) are a critical component for sustaining slow-cycling or quiescent HSCs. Interaction of HSCs with OBs through signaling and cell adhesion molecules maintains the balance in HSCs between cell division/proliferation and quiescence. In particular, the quiescent state is thought to be an essential mechanism to protect HSCs from stress and to sustain long-term hematopoiesis.

Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.

Adiponectin is a major insulin-sensitizing, multimeric hormone derived from adipose tissue that acts on muscle and liver to regulate whole-body glucose and lipid metabolism. Here, we describe a novel and highly conserved paralog of adiponectin designated as C1q/TNF-related protein (CTRP) 9. Of all the CTRP paralogs, CTRP9 shows the highest degree of amino acid identity to adiponectin in its globular C1q domain. CTRP9 is expressed predominantly in adipose tissue and females expresses higher levels of the transcript than males. Moreover, its expression levels in ob/ob mice changed in an age-dependent manner, with significant up-regulation in younger mice. CTRP9 is a secreted glycoprotein with multiple post-translational modifications in its collagen domain that include hydroxylated prolines and hydroxylated and glycosylated lysines. It is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum with levels varying according to sex and metabolic state of mice. Furthermore, CTRP9 and adiponectin can be secreted as heterooligomers when cotransfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally coimmunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, we show that adiponectin and CTRP9 form heterotrimers. In cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. Adenovirus-mediated overexpression of CTRP9 in obese (ob/ob) mice significantly lowered serum glucose levels. Collectively, these results suggest that CTRP9 is a novel adipokine, and further study of CTRP9 will yield novel mechanistic insights into its physiological and metabolic function.

OBJECTIVE

Obesity promotes macrophage infiltration into adipose tissue and is associated with increases in several cardiovascular diseases. Infusion of angiotensin II (AngII) to mice induces formation of abdominal aortic aneurysms (AAAs) with profound medial and adventitial macrophage infiltration. We sought to determine whether obesity promotes macrophage infiltration and proinflammatory cytokines in periaortic adipose tissue surrounding abdominal aortas and increases AngII-induced AAAs.

RESULTS

Hypertrophied white adipocytes surrounded abdominal aortas, whereas brown adipocytes surrounded thoracic aortas of obese mice. mRNA abundance of macrophage proinflammatory chemokines and their receptors were elevated with obesity to a greater extent in abdominal compared to thoracic periaortic adipose tissue. Periaortic adipose tissue explants surrounding abdominal aortas of obese mice released greater concentrations of MCP-1 and promoted more macrophage migration than explants from thoracic aortas. Male C57BL/6 mice were fed a high-fat (HF) diet for 1, 2, or 4 months and then infused with AngII (1000 ng/kg/min) for 28 days. AAA incidence increased progressively with the duration of HF feeding (18%, 36%,and 60%, respectively). Similarly, AngII-infused ob/ob mice exhibited increased AAAs compared to lean controls (76% compared to 32%, respectively, P<0.05). Infusion of AngII to obese mice promoted further macrophage infiltration into periaortic and visceral adipose tissue, and obese mice exhibiting AAAs had greater macrophage content in visceral adipose tissue than mice not developing AAAs.

CONCLUSIONS

Increased macrophage accumulation in periaortic adipose tissue surrounding abdominal aortas of AngII-infused obese mice is associated with enhanced AAA formation.

Adipose tissue growth has been proposed to involve recruitment of new blood vessels. Here, we test the hypothesis that delivery of an angiogenesis inhibitor in mice may prevent diet-induced obesity, the most common type of obesity in humans. We show that systemic administration of a selective angiogenesis inhibitor, TNP-470 (AGM-1470), prevents obesity in high caloric diet-fed wt mice as well as in genetically leptin-deficient ob/ob mice. Inhibition of obesity in mice by TNP-470 involves a reduction of vascularity in the adipose tissue. This therapeutic strategy appears to selectively affect the growth of adipose tissue as measured by the ratio between total fat and lean body mass. Interestingly, the treatment with TNP-470 results in decreased serum levels of low-density lipoprotein cholesterol. Furthermore, insulin levels are reduced, which indicates increased insulin sensitivity, suggesting that angiogenesis inhibitors may prevent the development of type II diabetes. Our findings suggest that similarly to growth and organogenesis in other tissues, adipose tissue growth is dependent on angiogenesis. Our observations may have conceptual implications for the prevention of obesity and related disorders.

Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of RPA32 and the entire RPA14 subunit. The structure revealed that RPA14 and the central part of RPA32 are structural homologues. Each subunit contains a central OB-fold domain, which also resembles the DNA-binding domains in RPA70; an N-terminal extension that interacts with the central OB-fold domain; and a C-terminal helix that mediate heterodimerization via a helix-helix interaction. The OB-fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA-binding domains, supporting a DNA-binding role for RPA32. The discovery of a third and fourth OB-fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB-folds, is conserved in evolution. The structure also suggests a mechanism for RPA trimer formation.

Peptide YY (PYY) and glucagon like peptide (GLP)-1 are cosecreted from intestinal L cells, and plasma levels of both hormones rise after a meal. Peripheral administration of PYY(3-36) and GLP-1(7-36) inhibit food intake when administered alone. However, their combined effects on appetite are unknown. We studied the effects of peripheral coadministration of PYY(3-36) with GLP-1(7-36) in rodents and man. Whereas high-dose PYY(3-36) (100 nmol/kg) and high-dose GLP-1(7-36) (100 nmol/kg) inhibited feeding individually, their combination led to significantly greater feeding inhibition. Additive inhibition of feeding was also observed in the genetic obese models, ob/ob and db/db mice. At low doses of PYY(3-36) (1 nmol/kg) and GLP-1(7-36) (10 nmol/kg), which alone had no effect on food intake, coadministration led to significant reduction in food intake. To investigate potential mechanisms, c-fos immunoreactivity was quantified in the hypothalamus and brain stem. In the hypothalamic arcuate nucleus, no changes were observed after low-dose PYY(3-36) or GLP-1(7-36) individually, but there were significantly more fos-positive neurons after coadministration. In contrast, there was no evidence of additive fos-stimulation in the brain stem. Finally, we coadministered PYY(3-36) and GLP-1(7-36) in man. Ten lean fasted volunteers received 120-min infusions of saline, GLP-1(7-36) (0.4 pmol/kg.min), PYY(3-36) (0.4 pmol/kg.min), and PYY(3-36) (0.4 pmol/kg.min) + GLP-1(7-36) (0.4 pmol/kg.min) on four separate days. Energy intake from a buffet meal after combined PYY(3-36) + GLP-1(7-36) treatment was reduced by 27% and was significantly lower than that after either treatment alone. Thus, PYY(3-36) and GLP-1(7-36), cosecreted after a meal, may inhibit food intake additively.

Obesity and type 2 diabetes are related metabolic disorders of high prevalence. The constitutive androstane receptor (CAR) was initially characterized as a xenobiotic receptor regulating the responses of mammals to xenotoxicants. In this study, we have uncovered an unexpected role of CAR in preventing obesity and alleviating type 2 diabetes. Using a high fat diet (HFD)-induced obesity model, we showed that treatment of wild type mice with the CAR agonist 1,4-bis[2-(3,5 dichloropyridyloxy)] benzene (TCPOBOP) efficiently prevented obesity from happening or reversed preinduced obesity. Treatment with TCPOBOP improved insulin sensitivity in both the HFD-induced type 2 diabetic model and the ob/ob mice. In contrast, CAR null mice maintained on a chow diet showed spontaneous insulin insensitivity, which cannot be relieved by TOPOBOP treatment. The hepatic steatosis in HFD-treated mice and ob/ob mice was markedly reduced by the TCPOBOP treatment. The metabolic benefits of CAR activation may have resulted from the combined effect of inhibition of lipogenesis, very low density lipoprotein secretion and export of triglycerides, and gluconeogenesis as well as increases in brown adipose tissue energy expenditure and peripheral fat mobilization. Moreover, the skeletal muscle of CAR-activated mice showed a decreased incomplete oxidation, despite having a lower expression level of peroxisome proliferator-activated receptor alpha and its target genes involved in fatty acid oxidation. In summary, our results have revealed an important metabolic function of CAR and may establish this "xenobiotic receptor" as a novel therapeutic target for the prevention and treatment of obesity and type 2 diabetes.

Neural stem cells (NSCs) in the adult brain continuously supply new neurons to the hippocampal dentate gyrus (DG) and the olfactory bulb (OB). Recent studies indicate that the progression from neural precursor cells (NPCs) to mature neurons is tightly controlled by coordinate cell-intrinsic programs and external signals within the neurogenic niche. In this review, we summarize both classes of regulatory factors involved in distinct stages of adult neurogenesis, including proliferation and lineage differentiation of NSCs, migration of neuroblasts and integration of newborn neurons. A full understanding of the wide variety of signaling pathways will ultimately provide precise targets for therapeutic applications.

OBJECTIVE

Fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) or n-3 PUFAs have been shown to reduce the incidence of coronary heart disease. Here we investigated the effect of highly purified eicosapentaenoic acid (EPA) on production of adiponectin, the only established antiatherogenic and antiinflammatory adipocytokine, in rodent models of obesity and human obese subjects.

RESULTS

We demonstrated that EPA increases adiponectin secretion in genetically obese ob/ob mice and high-fat diet-induced obese mice. In the in vitro coculture of adipocytes and macrophages, EPA reversed the coculture-induced decrease in adiponectin secretion at least in part through downregulation of tumor necrosis factor-alpha in macrophages. We also showed significant increase in plasma adiponectin concentrations in human obese subjects after a 3-month treatment with EPA (1.8 g daily). Multivariate regression analysis revealed that EPA treatment is the only independent determinant of plasma adiponectin concentrations.

CONCLUSIONS

This study demonstrates that EPA increases adiponectin secretion in rodent models of obesity and human obese subjects, possibly through the improvement of the inflammatory changes in obese adipose tissue. Because EPA has reduced the risk of major coronary events in a large-scale, prospective, randomized clinical trial, this study provides important insight into its therapeutic implication in obesity-related metabolic sequelae.

Thiazolidinediones have been shown to up-regulate adiponectin expression in white adipose tissue and plasma adiponectin levels, and these up-regulations have been proposed to be a major mechanism of the thiazolidinedione-induced amelioration of insulin resistance linked to obesity. To test this hypothesis, we generated adiponectin knock-out (adipo-/-) ob/ob mice with a C57B/6 background. After 14 days of 10 mg/kg pioglitazone, the insulin resistance and diabetes of ob/ob mice were significantly improved in association with significant up-regulation of serum adiponectin levels. Amelioration of insulin resistance in ob/ob mice was attributed to decreased glucose production and increased AMP-activated protein kinase in the liver but not to increased glucose uptake in skeletal muscle. In contrast, insulin resistance and diabetes were not improved in adipo-/-ob/ob mice. After 14 days of 30 mg/kg pioglitazone, insulin resistance and diabetes of ob/ob mice were again significantly ameliorated, which was attributed not only to decreased glucose production in the liver but also to increased glucose uptake in skeletal muscle. Interestingly, adipo-/-ob/ob mice also displayed significant amelioration of insulin resistance and diabetes, which was attributed to increased glucose uptake in skeletal muscle but not to decreased glucose production in the liver. The serum-free fatty acid and triglyceride levels as well as adipocyte sizes in ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were significantly reduced to a similar degree after 30 mg/kg pioglitazone. Moreover, the expressions of TNFalpha and resistin in adipose tissues of ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were decreased after 30 mg/kg pioglitazone. Thus, pioglitazone-induced amelioration of insulin resistance and diabetes may occur adiponectin dependently in the liver and adiponectin independently in skeletal muscle.

In this study, we have determined the effect of the divalent strontium salt S12911 on bone cell replication and bone formation in two culture systems. In the first series of experiments, half-calvariae of newborn rats were cultured with S12911 from 24 to 96 h and labeled with 3H-thymidine for the last 6 h of culture or treated with S12911 for 24 h and labeled for 24 h with 3H-proline 24-48 h after the removal of the agent. Calvariae were then processed for histomorphometry. S12911 at 10(-3) M increased the replication of preosteoblastic cells by 30-50% after 24 h and by 60% after 96 h of treatment. This effect was specific, since the number of labeled osteoblasts and of periosteal cells was not changed. A transient 24 h treatment with S12911 at 10(-3) M increased bone formation 24 and 48 h after the removal of the agent. 3H-proline labeled surfaces and bone formation rates were increased by 20%-35%. In the second series of experiments, sequential collagenase digestions were used to isolate cell populations enriched in fibroblasts or osteoblasts (Ob) from 22 day fetal rat calvariae. Treatment with S12911 at 10(-3) M for 24 h enhanced DNA synthesis by three- to fourfold in cell populations enriched in fibroblasts and preosteoblastic cells. The effect was less pronounced and inconsistent in Ob cells. S12911 at 10(-3) M for 24 h also increased collagen and non-collagen protein synthesis by 35% in Ob cells. These data indicate that the divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro, an effect that may contribute to the previously reported effects of S12911 on trabecular bone mass in vivo.

The adipose hormone leptin and its receptor are important for regulation of food intake and energy metabolism. Leptin also is involved in the growth of different tissues. In this study, we show the expression of leptin in primary cultures of normal human osteoblasts (hOBs) as evidenced by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Release of leptin into the medium also was found. Leptin was not detected in commercially available hOBs (NHOst) or in three different human monoclonal osteosarcoma cell lines. Leptin expression was observed in OBs in the mineralization and/or the osteocyte transition period but not during the matrix maturation period. Furthermore, hOBs and osteosarcoma cell lines expressed the long signal-transducing form of the leptin receptor (OB-Rb) as shown by RT-PCR. We observed no significant changes in leptin or OB-Rb genes in hOBs after incubation with recombinant leptin, indicating no autoregulation of the leptin expression. Incubation of both hOBs entering the mineralization phase and osteosarcoma cell lines with recombinant leptin markedly increased the number of mineralized nodules as shown by alizarin S staining. These findings indicate that leptin may be of importance for osteoblastic cell growth and bone mineralization.

OBJECTIVE

To determine if maintenance rituximab (MR) after standard chemotherapy improves progression-free survival (PFS) in advanced-stage indolent lymphoma.

METHODS

Patients with stage III-IV indolent lymphoma with responding or stable disease after cyclophosphamide, vincristine, and prednisone (CVP) chemotherapy were stratified by initial tumor burden, residual disease after CVP (minimal or gross), and histology, and randomly assigned to observation (OBS) or MR 375 mg/m(2) once per week for 4 weeks every 6 months for 2 years. PFS was the primary end point.

RESULTS

Three hundred eleven (282 with follicular lymphoma) evaluable patients who received CVP were randomly assigned to OBS (n = 158) or MR (n = 153). Best response improved in 22% MR versus 7% OBS patients (P = .00006). Toxicity was minimal in both study arms. Three-year PFS after random assignment was 68% MR versus 33% OBS (hazard ratio [HR] = 0.4; P = 4.4 x 10(-10) [all patients]) and 64% MR v 33% OBS (HR = 0.4; P = 9.2 x 10(-8) [patients with follicular lymphoma]). There was an advantage for MR regardless of Follicular Lymphoma International Prognostic Index score, tumor burden, residual disease, or histology. In multivariate analysis of MR patients, minimal disease after CVP was a favorable prognostic factor. OS at 3 years was 92% MR versus 86% OBS (HR = 0.6; log-rank one-sided P = .05) and, among patients with follicular lymphoma, OS was 91% MR versus 86% (HR = 0.6; log-rank one-sided P = .08). A trend favoring MR was observed among patients with high tumor burden (log-rank one-sided P = .03).

CONCLUSIONS

The E1496 study provides the first phase III data in untreated indolent lymphoma that MR after chemotherapy significantly prolongs PFS.

OBJECTIVE

To investigate leptin's effect on cartilage metabolism and the pathophysiology of osteoarthritis (OA).

METHODS

Messenger RNA (mRNA) expression and protein levels of leptin and leptin's receptor isoforms were measured by real-time reverse transcription-PCR and Western blot in osteoarthritic and normal cartilage. Osteoarthritic cartilage samples were obtained from two locations of the knee (n=11) and hip (n=6); from the main defective area (advanced OA) and from adjacent macroscopically and histological intact regions (minimal OA). Paired serum and synovial fluid (SF) leptin levels were measured. The effect of leptin was evaluated on chondrocyte proliferation, IL-1beta (interleukin-1beta), NO and metalloproteinases 9 and 13 (MMP-9, MMP-13) protein expression.

RESULTS

Leptin's and leptin's receptor (Ob-Rb) expression levels were significantly increased in advanced OA cartilage compared to minimal. Leptin was significantly increased in SF than serum samples. Also, leptin had a detrimental effect on chondrocyte proliferation and induced IL-1beta production and MMP-9 and MMP-13 protein expression. Furthermore, leptin's mRNA expression in advanced OA cartilage was significantly correlated with BMI of the patients.

CONCLUSIONS

The increased leptin levels in SF point toward a local effect of leptin in articular cartilage, while the observed intrajoint differences of leptin and Ob-Rb mRNA expression may be related to the grade of cartilage destruction. The observed production of IL-1beta, MMP-9 and MMP-13 by chondrocytes after leptin treatment indicates a pro-inflammatory and catabolic role of leptin on cartilage metabolism. Furthermore, the observed correlation of leptin's mRNA expression with BMI suggests that leptin may be a metabolic link between obesity and OA.

Leptin is induced by lipopolysaccharide (LPS) and cytokines. We investigated the role of leptin in LPS-induced toxicity using leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice. Sensitivity to LPS-induced mortality is significantly greater in ob/ob mice compared with their own lean littermates but not in db/db mice. LPS reduced serum glucose in both ob/ob and db/db mice but induced corticosterone only in db/db mice. Despite the very high basal levels of serum leptin in db/db mice, a twofold increase in serum leptin levels was observed after LPS in both db/db mice and their lean littermates. No differences were detected in LPS-induced serum levels of interleukin (IL)-1beta, tumor necrosis factor, macrophage inflammatory protein-1alpha, and interferon-gamma in ob/ob mice compared with their own littermates. In contrast, a blunted induction of IL-10 and IL-1 receptor antagonist (IL-1Ra) was observed in ob/ob mice compared with their littermates. In vitro, leptin induced IL-1Ra production and upregulated the IL-1Ra induction by LPS in macrophages. Moreover, treatment with leptin reversed the increased sensitivity to LPS-induced lethality found in ob/ob mice. These results suggest that leptin participates in the host response to inflammation by modulating the host immune and cytokine responses after LPS.

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.

The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1(ob)(-/-) mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1(ob)(-/-) mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1(ob)(-/-) mice without affecting other organ weight, while a high-protein diet overcame Atf4(-/-) and Rsk2(-/-) mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development.

The human antimicrobial peptide LL-37 plays an important role in host defense against infection. In addition to its antimicrobial action, other activities have been described in eukaryotic cells that may contribute to the healing response. In this study, we demonstrated that in vitro human cathelicidin activates migration of the human keratinocyte cell line HaCaT, involving phenotypic changes related to actin dynamics and associated to augmented tyrosine phosphorylation of proteins involved in focal adhesion complexes, such as focal adhesion kinase and paxillin. Other events involved in the LL-37 response were the induction of the Snail and Slug transcription factors, activation of matrix metalloproteinases and activation of the mitogen-activated protein kinase , and phosphoinositide 3-kinase/Akt signaling pathways. These signaling events could be mediated not only through the transactivation of EGFR but also through the induction of G-protein-coupled receptor FPRL-1 expression in these cells. Finally, by in vivo adenoviral transfer of the antimicrobial peptide to excisional wounds in ob/ob mice, we demonstrated that LL-37 significantly improved re-epithelialization and granulation tissue formation. The protective and regenerative activities of LL-37 support its therapeutic potential to promote wound healing.

The Wnt family of secreted signaling molecules has profound effects on diverse developmental processes, including the fate of mesenchymal progenitors. While activation of Wnt signaling blocks adipogenesis, inhibition of endogenous Wnt/beta-catenin signaling by Wnt10b promotes spontaneous preadipocyte differentiation. Transgenic mice with expression of Wnt10b from the FABP4 promoter (FABP4-Wnt10b) have less adipose tissue when maintained on a normal chow diet and are resistant to diet-induced obesity. Here we demonstrate that FABP4-Wnt10b mice largely avert weight gain and metabolic abnormalities associated with genetic obesity. FABP4-Wnt10b mice do not gain significant body weight on the ob/ob background, and at 8 weeks of age, they have an approximately 70% reduction in visceral and subcutaneous adipose tissues compared with ob/ob mice. Similarly, on the lethal yellow agouti (A(y)) background, FABP4-Wnt10b mice have 50-70% less adipose tissue weight and circulating leptin at 5 months of age. Wnt10b-Ay mice are more glucose tolerant and insulin sensitive than A(y) controls, perhaps due to reduced expression and circulation of resistin. Reduced expression of inflammatory cytokines may also contribute to improved glucose homeostasis.

In the olfactory bulb (OB) of zebrafish and other species, odors evoke fast oscillatory population activity and specific firing rate patterns across mitral cells (MCs). This activity evolves over a few hundred milliseconds from the onset of the odor stimulus. Action potentials of odor-specific MC subsets phase-lock to the oscillation, defining small and distributed ensembles within the MC population output. We found that oscillatory field potentials in the zebrafish OB propagate across the OB in waves. Phase-locked MC action potentials, however, were synchronized without a time lag. Firing rate patterns across MCs analyzed with low temporal resolution were informative about odor identity. When the sensitivity for phase-locked spiking was increased, activity patterns became progressively more informative about odor category. Hence, information about complementary stimulus features is conveyed simultaneously by the same population of neurons and can be retrieved selectively by biologically plausible mechanisms, indicating that seemingly alternative coding strategies operating on different time scales may coexist.

We studied the effects of genetic background on the phenotype of ob/ob mice, a model of severe obesity, insulin resistance, and diabetes caused by leptin deficiency. Despite a comparable degree of obesity and hyperinsulinemia, C57BL/6J ob/ob mice had much milder hyperglycemia and, surprisingly, normal circulating adiponectin levels despite still-prominent signs of insulin resistance. Hyperinsulinemic-euglycemic clamp revealed relatively less whole-body and muscle insulin resistance in C57BL/6J ob/ob mice, whereas liver insulin resistance tended to be more severe than in FVB/N ob/ob mice. C57BL/6J ob/ob mice had also more rapid clearance of circulating triglycerides and more severe hepatic steatosis. We suggest that strain-related distinction in lipid handling is the most important player in the differences in diabetic phenotype and insulin sensitivity, whereas the impact of circulating adiponectin levels on the overall phenotype of ob/ob mice is less important.