Christian Bjørbæk

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. J. Comp. Neurol. 395:535–547, 1998.

Identification of SOCS-3 as a Potential Mediator of Central Leptin Resistance

Leptin affects food intake and body weight by actions on the hypothalamus. Although leptin resistance is common in obesity, mechanisms have not been identified. We examined the effect of leptin on expression of the suppressors-of-cytokine-signaling (SOCS) family of proteins. Peripheral leptin administration to ob/ob, but not db/db mice, rapidly induced SOCS-3 mRNA in hypothalamus, but had no effect on CIS, SOCS-1, or SOCS-2. A leptin-dependent increase of SOCS-3 mRNA was seen in areas of hypothalamus expressing high levels of the leptin receptor long form. In mammalian cell lines, SOCS-3, but not CIS or SOCS-2, blocked leptin-induced signal transduction. Expression of SOCS-3 mRNA in the arcuate and dorsomedial hypothalamic nuclei is increased in Ay/a mice, a model of leptin-resistant murine obesity. In conclusion, SOCS-3 is a leptin-inducible inhibitor of leptin signaling, and a potential mediator of leptin resistance in obesity.

Divergent signaling capacities of the long and short isoforms of the leptin receptor.

Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs) that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo.

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.

Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity

Obesity in humans and in rodents is usually associated with high circulating leptin levels and leptin resistance. To examine the molecular basis for leptin resistance, we determined the ability of leptin to induce hypothalamic STAT3 (signal transducer and activator of transcription) signaling in C57BL/6J mice fed either low-fat or high-fat diets. In mice fed the low-fat diet, leptin activated STAT3 signaling when administered via the intraperitoneal (ip) or the intracerebroventricular (icv) route, with the half-maximal dose being 30-fold less when given by the icv route. The high-fat diet increased body-weight gain and plasma leptin levels. After 4 weeks on the diet, hypothalamic STAT3 signaling after ip leptin administration was equivalent in both diet groups. In contrast, peripherally administered leptin was completely unable to activate hypothalamic STAT3 signaling, as measured by gel shift assay after 15 weeks of high-fat diet. Despite the absence of detectable signaling after peripheral leptin at 15 weeks, the mice fed the high-fat diet retained the capacity to respond to icv leptin, although the magnitude of STAT3 activation was substantially reduced. These results suggest that leptin resistance induced by a high-fat diet evolves during the course of the diet and has at least two independent causes: an apparent defect in access to sites of action in the hypothalamus that markedly limits the ability of peripheral leptin to activate hypothalamic STAT signaling, and an intracellular signaling defect in leptin-responsive hypothalamic neurons that lies upstream of STAT3 activation.

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 by125I-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.

Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3

Leptin is an adipocyte-derived hormone that regulates energy balance and neuroendocrine function primarily by acting on specific hypothalamic pathways. Resistance to the weight reducing effects of leptin is a feature of most cases of human and rodent obesity, yet the molecular basis of leptin resistance is poorly understood. We have previously identified suppressor of cytokine signaling-3 (Socs3) as a leptin-induced negative regulator of leptin receptor signaling and potential mediator of leptin resistance. However, due to the non-viability of mice with targeted disruption of Socs3 (ref. 6), the importance of Socs3 in leptin action in vivo was unclear. To determine the functional significance of Socs3 in energy balance in vivo we undertook studies in mice with heterozygous Socs3 deficiency (Socs3+/−). We report here that Socs3+/− mice display greater leptin sensitivity than wild-type control mice: Socs3+/− mice show both enhanced weight loss and increased hypothalamic leptin receptor signaling in response to exogenous leptin administration. Furthermore, Socs3+/− mice are significantly protected against the development of diet-induced obesity and associated metabolic complications. The level of Socs3 expression is thus a critical determinant of leptin sensitivity and obesity susceptibility in vivo and this molecule is a potential target for therapeutic intervention.

Divergent Roles of SHP-2 in ERK Activation by Leptin Receptors

The protein tyrosine phosphatase SHP-2 has been proposed to serve as a regulator of leptin signaling, but its specific roles are not fully examined. To directly investigate the role of SHP-2, we employed dominant negative strategies in transfected cells. We show that a catalytically inactive mutant of SHP-2 blocks leptin-stimulated ERK phosphorylation by the long leptin receptor, ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially inhibits ERK phosphorylation. We find similar effects of the SHP-2 mutants after examining stimulation of an ERK-dependentegr-1 promoter-construct by leptin. We also demonstrate ERK phosphorylation and egr-1 mRNA expression in the hypothalamus by leptin. Analysis of signaling by ObRb lacking intracellular tyrosine residues or by the short leptin receptor, ObRa, enabled us to conclude that two pathways are critical for ERK activation. One pathway does not require the intracellular domain of ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is required for both pathways, whereas activation of ERK via Tyr-985 of ObRb also requires tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator of ERK by leptin receptors, and both the adaptor function and the phosphatase activity of SHP-2 are critical for this regulation.

Aminoacid polymorphisms of insulin receptor substrate-1 in non-insulin-dependent diabetes mellitus.

Since relative or absolute insulin deficiency and insulin insensitivity are involved in the aetiology of non-insulin-dependent diabetes mellitus (NIDDM), we examined whether patients with NIDDM exhibit genetic variability in the coding region of insulin receptor substrate-1 (IRS-1), a candidate gene that is ubiquitous in insulin-sensitive and insulin-like growth factor 1 (IGF1) sensitive tissues, including those that determine glucose production and clearance and those with regulatory effects on pancreatic beta-cell function. IRS-1 has a central role as an adaptor molecule that links the insulin-receptor and IGF1-receptor kinases with enzymes that regulate cellular metabolism and growth. Single-stranded conformation polymorphism analysis and direct nucleotide sequencing were applied to genomic DNA from 86 unrelated patients with NIDDM and 76 normoglycaemic controls. 10 of the patients with NIDDM and 3 of the controls were heterozygous at codon 972 for a polymorphism in which glycine was substituted with arginine. Moreover, at codon 513, 6 patients with NIDDM and 2 controls had a heterozygous polymorphism with a transition from alanine to proline. None of the polymorphism carriers had both aminoacid variants and the total allelic frequency of IRS-1 polymorphisms was about three times higher in patients with NIDDM than in controls (p = 0.02). Both aminoacid substitutions were located close to tyrosine phosphorylation motifs that are putative recognition sites for insulin and IGF1 signal transmission proteins. Analysis of the phenotypes showed that patients with NIDDM who had IRS-1 variants did not differ in their degree of insulin resistance compared with patients without known IRS-1 polymorphisms. However, carriers of the codon 972 variant had significantly lower plasma levels of fasting insulin and C-peptide. Our results suggest that aminoacid polymorphisms in IRS-1 may be involved in the aetiology of a subset of late-onset NIDDM.

Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptins effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.