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Biomedical subjects

J S Flier

Publications and source records attributed to J S Flier.

At least 37 records · Page 2Linked to original sources
PubMed2000

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

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

Animals↗
PubMed2000

In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin.

To determine whether leptin signal transduction is exerted directly upon insulin-sensitive tissues in vivo, we examined the ability of iv leptin to acutely stimulate phosphorylation of STAT3, STAT1, and MAPK, and activities of PI 3-kinase and Akt, in insulin-sensitive tissues of normal rats. Both leptin (1 mg/kg iv x 3 min) and insulin (10 U/kg iv x 3 min) stimulated tyrosine phosphorylation of STAT3 5.6- to 6.0-fold and of STAT1 4.0-fold in adipose tissue. Leptin tended to increase STAT3 phosphorylation in liver and muscle. Both hormones also increased MAPK phosphorylation: leptin increased it 3.2- to 3.8-fold in adipose tissue and liver, whereas insulin stimulated MAPK phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and 2.5-fold in muscle. Leptin was much less effective than insulin at stimulating IRS pathways. Leptin increased IRS-1-associated PI 3-kinase activity in adipose tissue only 2.0-fold (P < 0.01) compared with the 10-fold effect of insulin. IRS-2-associated PI 3-kinase activity was increased 1.7-fold (P < 0.01) by leptin in liver and 6-fold by insulin. Akt phosphorylation and activity were not changed by leptin but increased with insulin. Lower concentrations of leptin (10 and 50 microg/kg) also stimulated STAT3 phosphorylation in fat. These effects appear to be direct because 3 min after leptin intracerebroventricular injection, phosphorylation of STAT3, STAT1, and MAPK were not stimulated in hypothalamus or adipose tissue. Furthermore, leptin activated STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes. Leptin did not activate STAT3 or MAPK in adipose tissue of db/db mice. Thus, leptin rapidly activates signaling pathways directly at the level of insulin sensitive tissues through the long-form leptin receptor, and these pathways overlap with, but are distinct from, those engaged by insulin.

3T3 Cells↗
PubMed1999

Cardiovascular abnormalities in transgenic mice with reduced brown fat: an animal model of human obesity.

BACKGROUND: A new model of murine obesity has recently been developed through transgenic ablation of brown adipose tissue that manifests typical metabolic complications of obesity, including insulin resistance and non-insulin-dependent diabetes mellitus. The cardiovascular phenotype has not been defined. METHODS AND RESULTS: Transthoracic echocardiography, aortic catheterization, isolated whole-heart studies, and morphometric histology defined cardiac structure and function in 30 transgenic mice with reduced brown fat and 30 matched wild-type controls. Obesity was indicated by a 77% increase in body weight and was accompanied by elevated systemic pressures (mean aortic blood pressure 85+/-1 versus 66+/-2 mm Hg; P<0.01), left ventricular dilation and hypertrophy (mass/body weight 4.0+/-0.2 versus 2.7+/-0.3 mg/g; P<0.01), and high cardiac output (cardiac index 3.2+/-0.4 versus 2.4+/-0.1 mL x kg(-1) x min(-1); P<0.01). Baseline functional parameters assessed in vitro were not different, but after imposition of zero-flow ischemia, significant relaxation impairment developed in obese mice. Although morphometrically determined myocyte diameters were similar, the percentage of interstitial fibrosis was significantly increased in transgenic mice compared with wild-type controls (7.5+/-2% versus 4. 2+/-0.2%; P<0.01). CONCLUSIONS: Transgenic ablation of brown adipose tissue is associated not only with obesity but also with systemic hypertension, left ventricular hypertrophy with eccentric remodeling and fibrosis, and high cardiac output, a unique constellation of findings strikingly similar to that seen in human obesity. Mice with reduced brown fat may serve as a new model for the cardiovascular morbid complications associated with obesity in humans.

Adipose Tissue, Brown↗
PubMed1999

The role of SOCS-3 in leptin signaling and leptin resistance.

We earlier demonstrated that leptin induces expression of SOCS-3 mRNA in the hypothalamus. Furthermore, transfection data suggest that SOCS-3 is an inhibitor of leptin signaling. However, little is known about the regulation of SOCS-3 expression by leptin and the mechanism by which SOCS-3 inhibits leptin action. We here show that in CHO cells stably expressing the long form of the leptin receptor (CHO-OBRl), leptin induces transient expression of endogenous SOCS-3 mRNA but not of CIS, SOCS-1, or SOCS-2 mRNA. SOCS-3 protein levels were maximal after 2-3 h of leptin treatment and remained elevated at 20 h. Furthermore, in leptin-pretreated CHO-OBRl cells, proximal leptin signaling was blocked for more than 20 h after pretreatment, thus correlating with increased SOCS-3 expression. Leptin pretreatment did not affect cell surface expression of leptin receptors as measured by (125)I-leptin binding assays. In transfected COS cells, forced expression of SOCS-3 results in inhibition of leptin-induced tyrosine phosphorylation of JAK2. Finally, JAK2 co-immunoprecipitates with SOCS-3 in lysates from leptin-treated COS cells. These results suggest that SOCS-3 is a leptin-regulated inhibitor of proximal leptin signaling in vivo. Excessive SOCS-3 activity in leptin-responsive cells is therefore a potential mechanism for leptin resistance, a characteristic feature in human obesity.

Animals↗
PubMed1999

Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area.

Recent studies have reinforced the view that the lateral hypothalamic area (LHA) regulates food intake and body weight. We identified leptin-sensitive neurons in the arcuate nucleus of the hypothalamus (Arc) that innervate the LHA using retrograde tracing with leptin administration. We found that retrogradely labeled cells in the Arc contained neuropeptide Y (NPY) mRNA or proopiomelanocortin (POMC) mRNA. Following leptin administration, NPY cells in the Arc did not express Fos but expressed suppressor of cytokine signaling-3 (SOCS-3) mRNA. In contrast, leptin induced both Fos and SOCS-3 expression in POMC neurons, many of which also innervated the LHA. These findings suggest that leptin directly and differentially engages NPY and POMC neurons that project to the LHA, linking circulating leptin and neurons that regulate feeding behavior and body weight homeostasis.

Animals↗
PubMed1999

Distinct physiologic and neuronal responses to decreased leptin and mild hyperleptinemia.

Leptin acts on specific populations of hypothalamic neurons to regulate feeding behavior, energy expenditure, and neuroendocrine function. It is not known, however, whether the same neural circuits mediate leptin action across its full biologic dose-response curve, which extends over a broad range, from low levels seen during starvation to high levels characteristic of obesity. Here, we show that the characteristic fall in leptin with fasting causes a rise in neuropeptide Y (NPY) messenger RNA (mRNA), as well as a fall in POMC and cocaine and amphetamine-regulated transcript (CART) mRNAs. Sc infusion of leptin sufficient to maintain plasma levels within the physiologic range during the fast prevents changes in the expression of these peptides, as well as changes in neuroendocrine function, demonstrating that multiple neural circuits are highly sensitive to small changes in leptin within its low physiologic range. In contrast, a modest elevation of plasma leptin above the normal fed range by constant sc infusion, which produced marked reduction in food intake and body weight, decreased NPY mRNA in the arcuate hypothalamic nucleus but did not affect the levels of mRNAs encoding the anorexigenic peptides alpha-MSH, CART or CRH. These results suggest that the dose response characteristics of leptin on hypothalamic target neurons at the level of mRNA expression are variable, with some neurons (e.g. NPY) responding across a broad dose range and others (e.g. POMC and CART) showing a limited response within the low range. These results further suggest that the central targets of leptin that mediate the transition from starvation to the fed state may be distinct from those that mediate the response to overfeeding and obesity.

Animals↗
PubMed1999

Activation of SOCS-3 messenger ribonucleic acid in the hypothalamus by ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a neurocytokine expressed in glial cells that acts on brain cells to promote gene expression, survival, and differentiation. When administered systemically, CNTF reduces food intake and body weight in rodents. Genes encoding suppressors of cytokine signaling (SOCS) are induced by cytokines that activate membrane receptors in the same class as those that are activated by CNTF. We therefore examined the ability of CNTF to induce expression of socs genes in brain and peripheral tissues of rats and mice. Peripheral CNTF administration to ob/ob mice rapidly induced SOCS-3 messenger RNA (mRNA) in hypothalamus, as determined by Northern blotting and quantitative RT-PCR, but had no effect on cytokine-inducible sequence (CIS), SOCS-1, or SOCS-2 mRNA. In situ hybridization histochemistry of hypothalamus from ob/ob mice and normal rats demonstrated that CNTF induced SOCS-3 mRNA in the arcuate nucleus (Arc). Strong hybridization signals were also detected in the ependymal lining of the ventricles and the subfornical organ. This hybridization pattern was distinct from that resulting from peripheral leptin treatment with overlapping hybridization patterns only in the Arc. CNTF also induced expression of CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA in the liver, and SOCS-2 and SOCS-3 mRNA in the kidney. CNTF induced SOCS-3 mRNA and SOCS-3 protein levels in an astrocyte cell line. Transient expression of SOCS-3, but not CIS or SOCS-2, inhibited CNTF-induced signal transduction in astrocytes. In conclusion, SOCS-3 mRNA is specifically induced by CNTF in regions of the hypothalamus that are both overlapping and distinct from that induced by leptin. Similar to leptin, the Arc is likely to be a direct target of CNTF, and this region may play a role in the body weight-reducing effects of CNTF. SOCS-3 is a negative regulator of CNTF signal transduction, and inhibitors of SOCS-3 function may enhance endogenous CNTF signaling after neuronal injury or enhance the body weight-reducing effect of CNTF after peripheral administration.

Animals↗
PubMed1999

Regulation of neuronal and glial proteins by leptin: implications for brain development.

The complete absence of leptin causes severe obesity in mice and humans, but its physiological roles are incompletely defined. Earlier studies reported decreased brain weight and impaired myelination in ob/ob and db/db mice. Here we have examined the effects of leptin deficiency and postnatal leptin treatment on brain weight, the expression of a broad array of neuronal and glial markers, and locomotor activity. ob/ob and db/db mice have reduced brain weight and an immature pattern of expression of synaptic and glial proteins, with growth-associated protein being elevated in the neocortex and hippocampus, and syntaxin-1, synaptosomal-associated protein-25, and synaptobrevin being decreased. The expression of myelin basic protein, proteolipid protein, and glial fibrillary acidic protein was also decreased in the neocortex, hippocampus, and striatum of ob/ob and db/db mice. Six weeks of leptin treatment initiated at week 4 increased brain weight and protein content, increased locomotor activity, and normalized levels of growth-associated protein, syntaxin-1, and synaptosomal-associated protein-25 in ob/ob mice without affecting synaptobrevin and glial proteins. In contrast with ob/ob and db/db mice, obese agouti (AY/a) mice had normal brain weight and expression of synaptic and glial proteins. These findings suggest that leptin, a peripheral signal of energy stores in adult animals, is required for normal neuronal and glial maturation in the mouse nervous system.

Animals↗
PubMed1999

Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation.

Long (ObRb) and short (ObRa) leptin receptor isoforms are thought to play essential roles in mediating leptin signaling and the transport and degradation of leptin, respectively. Although the capacity of these cloned receptor species to mediate signal transduction has been reported, there is no information on the ability of individual receptor species to mediate leptin internalization and degradation or to undergo ligand-induced downregulation. We therefore studied these parameters in Chinese hamster ovary (CHO) cells stably expressing either ObRa or ObRb isoforms of the leptin receptor. We determined that both ObRa and ObRb mediated internalization of 125I-labeled leptin by a temperature- and coated pit-dependent mechanism. Both ObRa and ObRb also mediated degradation of 125I-leptin by a lysosomal mechanism, and this was more efficiently mediated by ObRa in these cells. Neither leptin internalization nor degradation by ObRa was affected by mutation of the conserved Box 1 motif. By studying deletion mutants of ObRa, we found that efficient internalization was dependent on a motif located between amino acids 8 and 29 of the intracellular domain of ObRa. Exposure of cells expressing ObRa or ObRb to unlabeled leptin for 90 min at 37 degrees C produced downregulation of available surface receptors, and this effect was of greater magnitude in cells expressing ObRb. Whereas CHO cells expressing the growth hormone receptor showed marked downregulation of ligand binding after exposure to dexamethasone (DEX) or phorbol myristic acid (PMA), PMA had no effect on expression of ObRa or ObRb, and DEX reduced binding to cells expressing ObRb by 15%. Thus, the two leptin receptor isoforms, ObRa and ObRb, mediate leptin internalization by a coated pit-dependent mechanism, leptin degradation by a lysosomal pathway, and ligand-induced receptor downregulation. The differential capacity of the two receptor isoforms may relate to the different roles of the receptor isoforms in the biology of leptin.

Amino Acid Sequence↗
PubMed1998

Mice lacking melanin-concentrating hormone are hypophagic and lean.

Feeding is influenced by hypothalamic neuropeptides that promote (orexigenic peptides) or inhibit feeding. Of these, neuropeptide Y (NPY) in the arcuate nucleus and melanin-concentrating hormone (MCH) and orexins/hypocretins in the lateral hypothalamus have received attention because their expression is increased during fasting and because they promote feeding when administered centrally. Surprisingly, absence of the orexigenic neuropeptide NPY fails to alter feeding or body weight in normal mice. As deficiency of a single component of the pathway that limits food intake (such as leptin or receptors for melanocortin-4) causes obesity, it has been suggested that orexigenic signals are more redundant than those limiting food intake. To define further the physiological role of MCH and to test the redundancy of orexigenic signals, we generated mice carrying a targeted deletion of the MCH gene. MCH-deficient mice have reduced body weight and leanness due to hypophagia (reduced feeding) and an inappropriately increased metabolic rate, despite their reduced amounts of both leptin and arcuate nucleus pro-opiomelanocortin messenger RNA. Our results show that MCH is a critical regulator of feeding and energy balance which acts downstream of leptin and the melanocortin system, and that deletion of a gene encoding a single orexigenic peptide can result in leanness.

Animals↗
PubMed1998

Distributions of leptin receptor mRNA isoforms in the rat brain.

Leptin, secreted by white adipocytes, has profound feeding, metabolic, and neuroendocrine effects. Leptin acts on the brain, but the specific anatomic sites and pathways responsible for mediating these effects are still unclear. We have systematically examined distributions of mRNA of leptin receptor isoforms in the rat brain by using a probe specific for the long form and a probe recognizing all known forms of the leptin receptor. The mRNA for the long form of the receptor (OB-Rb) localized to selected nuclear groups in the rat brain. Within the hypothalamus, dense hybridization was observed in the arcuate, dorsomedial, ventromedial, and ventral premamillary nuclei. Within the dorsomedial nucleus, particularly intense hybridization was observed in the caudal regions of the nucleus ventral to the compact formation. Receptors were preferentially localized to the dorsomedial division of the ventromedial nucleus. Hybridization accumulated throughout the arcuate nucleus, extending from the retrochiasmatic region to the posterior periventricular region. Moderate hybridization was observed in the periventricular hypothalamic nucleus, lateral hypothalamic area, medial mammillary nucleus, posterior hypothalamic nucleus, nucleus of the lateral olfactory tract, and within substantia nigra pars compacta. Several thalamic nuclei were also found to contain dense hybridization. These groups included the mediodorsal, ventral anterior, ventral medial, submedial, ventral posterior, and lateral dorsal thalamic nuclei. Hybridization was also observed in the medial and lateral geniculate nuclei. Intense hybridization was observed in the Purkinje and granular cell layers of the cerebellum. A probe recognizing all known forms of the leptin receptor hybridized to all of these sites within the brain. In addition, intense hybridization was observed in the choroid plexus, meninges, and also surrounding blood vessels. These findings indicate that circulating leptin may act through hypothalamic nuclear groups involved in regulating feeding, body weight, and neuroendocrine function. The localization of leptin receptor mRNA in extrahypothalamic sites in the thalamus and cerebellum suggests that leptin may act on specific sensory and motor systems. Leptin receptors localized in nonneuronal cells in the meninges, choroid plexus, and blood vessels may be involved in transport of leptin into the brain and in the clearance of leptin from the cerebrospinal fluid.

Adipocytes↗
PubMed1998

Synchronicity of frequently sampled, 24-h concentrations of circulating leptin, luteinizing hormone, and estradiol in healthy women.

Leptin, an adipocyte hormone, is a trophic factor for the reproductive system; however, it is still unknown whether there is a dynamic relation between fluctuations in circulating leptin and hypothalamic-pituitary-ovarian (HPO) axis hormones. To test the hypothesis that fluctuations in plasma leptin concentrations are related to the levels of luteinizing hormone (LH) and estradiol, we sampled plasma from six healthy women every 7 min for 24 h during days 8-11 of the menstrual cycle. Cross-correlation analysis throughout the 24-h cycle revealed a relation between release patterns of leptin and LH, with a lag of 42-84 min but no significant cross-correlation between LH and estradiol. The ultradian fluctuations in leptin levels showed pattern synchrony with those of both LH and estradiol as determined by cross-approximate entropy (cross-ApEn). At night, as leptin levels rose to their peak, the pulsatility profiles of LH changed significantly and became synchronous with those of leptin. LH pulses were fewer, of longer duration, higher amplitude, and larger area than during the day. Moreover, the synchronicity of LH and leptin occurred late at night, at which time estradiol and leptin also exhibited significantly stronger pattern coupling than during the day. We propose that leptin may regulate the minute-to-minute oscillations in the levels of LH and estradiol, and that the nocturnal rise in leptin may determine the change in nocturnal LH profile in the mid-to-late follicular phase that precedes ovulation. This may explain the disruption of hypothalamic-pituitary-ovarian function that is characteristic of states of low leptin release, such as anorexia nervosa and cachexia.

Activity Cycles↗
PubMed1998

Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function.

Leptin is thought to regulate energy balance through effects on food intake and thermogenesis. In addition, leptin may serve as a mediator of the neuroendocrine response to starvation, and may modulate the stress response and the timing of puberty. A role for leptin in development is suggested by the presence of neuroendocrine and structural neuronal abnormalities in ob/ob mice with genetic leptin deficiency. Here, we sought to determine the ontogeny of leptin expression and its relationship to the developing neuroendocrine axis. Leptin increased 5-10-fold in female mice during the second postnatal week independent of fat mass, and declined after weaning. The rise in leptin preceded the establishment of adult levels of corticosterone, thyroxine, and estradiol. In contrast to adult mice, leptin was not acutely regulated by food deprivation during the early postnatal period. Circadian rhythms of leptin, corticosterone, and thyroxine were regulated by food intake in adult mice. When ad libitum feeding was restricted to the light cycle, peak corticosterone levels were shifted to the beginning of the light cycle and coincided with the nadir of leptin. The inverse relationship between leptin and corticosterone was maintained such that a rise in leptin after feeding was associated with a decline in corticosterone. To determine whether changes in corticosterone during food restriction are mediated by leptin, we compared the patterns of corticosterone levels among ob/ob, db/db, and lean mice. Despite their higher basal levels of corticosterone, leptin deficiency in ob/ ob mice did not prevent the nocturnal rise in corticosterone. In contrast, the nocturnal surge of corticosterone was blunted in db/db mice. Therefore, it is likely that factors in addition to leptin are involved in the regulation of the circadian rhythm of corticosterone. The temporal relationship between leptin and other hormones in neonatal and adult mice suggests that leptin is involved in the maturation and function of the neuroendocrine axis.

Animals↗