Leptin-regulated Genes Research Articles

Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition in Female Mice.

SummaryEpidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.

Transcriptional and physiological roles for STAT proteins in leptin action

ObjectivesLeptin acts via its receptor LepRb on specialized neurons in the brain to modulate food intake, energy expenditure, and body weight. LepRb activates signal transducers and activators of transcription (STATs, including STAT1, STAT3, and STAT5) to control gene expression. MethodsBecause STAT3 is crucial for physiologic leptin action, we used TRAP-seq to examine gene expression in LepRb neurons of mice ablated for Stat3 in LepRb neurons (Stat3LepRbKO mice), revealing the STAT3-dependent transcriptional targets of leptin. To understand roles for STAT proteins in leptin action, we also ablated STAT1 or STAT5 from LepRb neurons and expressed a constitutively-active STAT3 (CASTAT3) in LepRb neurons. ResultsWhile we also found increased Stat1 expression and STAT1-mediated transcription of leptin-regulated genes in Stat3LepRbKO mice, ablating Stat1 in LepRb neurons failed to alter energy balance (even on the Stat3LepRbKO background); ablating Stat5 in LepRb neurons also failed to alter energy balance. Importantly, expression of a constitutively-active STAT3 (CASTAT3) in LepRb neurons decreased food intake and body weight and improved metabolic parameters in leptin-deficient (ob/ob) mice, as well as in wild-type animals. ConclusionsThus, STAT3 represents the unique STAT protein required for leptin action and STAT3 suffices to mediate important components of leptin action in the absence of other LepRb signals.

Leptin Receptor as a Potential Target to Inhibit Human Testicular Seminoma Growth

Although in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the invivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer.

Ancient origins and evolutionary conservation of intracellular and neural signaling pathways engaged by the leptin receptor.

In mammals, leptin acts on leptin receptor (LepR) -expressing neurons in the brain to suppress food intake and stimulate whole-body metabolism. A similar action of leptin on food intake has been reported in the frog Xenopus laevis and in several bony fishes. However, the intracellular signaling and neural pathways by which leptin regulates energy balance have not been investigated outside of mammals. Using reporter assays and site-directed mutagenesis we show that the frog LepR signals via signal transducer and activator of transcription (STAT) 3 and STAT5 through evolutionarily conserved tyrosine residues in the LepR cytoplasmic domain. In situ hybridization histochemistry for LepR mRNA in brain and pituitary showed strong expression in the magno- and parvocellular divisions of the anterior preoptic area (homologous to the mammalian paraventricular nucleus), the suprachiasmatic nucleus, ventral hypothalamus, and pars intermedia and pars distalis of the anterior pituitary. Leptin injection increased phosphorylated STAT3 immunoreactivity in LepR mRNA-positive cells, and induced socs3 and pomc mRNAs. Microarray analysis of preoptic area/hypothalamus/pituitary 2 hours after leptin injection identified leptin-regulated genes that included c-fos, a known leptin-activated gene; pituitary follicle-stimulating hormone subunit β, suggesting an important role for leptin in the reproductive axis of frogs; and B-cell translocation factor 2, which has important functions in neurogenesis. Our findings support that the intracellular signaling pathways and neural substrates that mediate leptin actions on energy balance were present in the common ancestor of modern amphibians and amniotes and have been conserved over 350 million years of evolutionary time.

Regulation of insulin and leptin signaling by muscle suppressor of cytokine signaling 3 (SOCS3).

Skeletal muscle resistance to the key metabolic hormones, leptin and insulin, is an early defect in obesity. Suppressor of cytokine signaling 3 (SOCS3) is a major negative regulator of both leptin and insulin signaling, thereby implicating SOCS3 in the pathogenesis of obesity and associated metabolic abnormalities. Here, we demonstrate that SOCS3 mRNA expression is increased in murine skeletal muscle in the setting of diet-induced and genetic obesity, inflammation, and hyperlipidemia. To further evaluate the contribution of muscle SOCS3 to leptin and insulin resistance in obesity, we generated transgenic mice with muscle-specific overexpression of SOCS3 (MCK/SOCS3 mice). Despite similar body weight, MCK/SOCS3 mice develop impaired systemic and muscle-specific glucose homeostasis and insulin action based on glucose and insulin tolerance tests, hyperinsulinemic-euglycemic clamps, and insulin signaling studies. With regards to leptin action, MCK/SOCS3 mice exhibit suppressed basal and leptin-stimulated activity and phosphorylation of alpha2 AMP-activated protein kinase (α2AMPK) and its downstream target, acetyl-CoA carboxylase (ACC). Muscle SOCS3 overexpression also suppresses leptin-regulated genes involved in fatty acid oxidation and mitochondrial function. These studies demonstrate that SOC3 within skeletal muscle is a critical regulator of leptin and insulin action and that increased SOCS may mediate insulin and leptin resistance in obesity.

Leptin up‐regulates pro‐inflammatory cytokines in discrete cells within mouse colon

Dysregulation of leptin associated with obesity is implicated in obesity-related colon cancer, but mechanisms are elusive. Increased adiposity and elevated plasma leptin are associated with perturbed metabolism in colon and leptin receptors are expressed on colon epithelium. We hypothesise that obesity increases the sensitivity of the colon to cancer by disrupting leptin-regulated gene targets within colon tissues. PCR arrays were used to firstly identify leptin responsive genes and secondly to identify responses to leptin challenge in wild-type mice, or those lacking leptin (ob/ob). Leptin-regulated genes were localised in the colon using in situ hybridisation. IL6, IL1β and CXCL1 were up-regulated by leptin and localised to discrete cells in gut epithelium, lamina propria, muscularis and at the peritoneal serosal surface. Leptin regulates pro-inflammatory genes such as IL6, IL1β and CXCL1, and might increase the risk of colon cancer among obese individuals.

Protein Phosphatase 1 (PP-1)-Dependent Inhibition of Insulin Secretion by Leptin in INS-1 Pancreatic β-Cells and Human Pancreatic Islets

Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca²(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.

Antidiabetic Effects of IGFBP2, a Leptin-Regulated Gene

We tested whether leptin can ameliorate diabetes independent of weight loss by defining the lowest dose at which leptin treatment of ob/ob mice reduces plasma glucose and insulin concentration. We found that a leptin dose of 12.5 ng/hr significantly lowers blood glucose and that 25 ng/hr of leptin normalizes plasma glucose and insulin without significantly reducing body weight, establishing that leptin exerts its most potent effects on glucose metabolism. To find possible mediators of this effect, we profiled liver mRNA using microarrays and identified IGF Binding Protein 2 (IGFBP2) as being regulated by leptin with a similarly high potency. Overexpression of IGFBP2 by an adenovirus reversed diabetes in insulin-resistant ob/ob, Ay/a, and diet-induced obese mice, as well as insulin-deficient streptozotocin-treated mice. Hyperinsulinemic clamp studies showed a 3-fold improvement in hepatic insulin sensitivity following IGFBP2 treatment of ob/ob mice. These results show that IGFBP2 can regulate glucose metabolism, a finding with potential implications for the pathogenesis and treatment of diabetes.

Leptin-regulated gene expression in MCF-7 breast cancer cells: mechanistic insights into leptin-regulated mammary tumor growth and progression

Leptin-regulated gene expression in MCF-7 breast cancer cells: mechanistic insights into leptin-regulated mammary tumor growth and progression

Novel Leptin-Regulated Genes Revealed by Transcriptional Profiling of the Hypothalamic Paraventricular Nucleus

Leptin plays a major role in coordinating the integrated response of the CNS to changes in nutritional state. Neurons within the paraventricular nucleus (PVN) of the hypothalamus express leptin receptors and receive dense innervation from leptin receptor-expressing neurons in the arcuate nucleus. To obtain new insights into the effects of circulating leptin on PVN function, we compared global transcriptional profiles of laser-captured PVN from ad libitum fed mice versus 48 h fasted mice receiving either sham or leptin treatment intraperitoneally. Five hundred twenty-seven PVN-expressed genes were altered by fasting in a manner that was at least partially reversible by leptin. Consistent with previous reports, thyrotrophin releasing hormone mRNA levels were decreased by fasting but restored to fed levels with leptin treatment. mRNA levels of oxytocin, vasopressin, and somatostatin were also reduced by fasting and restored by leptin. Given the known effects of leptin on synaptic remodeling, it is notable that, among the top 15 genes that were positively regulated by leptin, five have been implicated in synaptic function and/or plasticity (basigin, apolipoprotein E, Gap43, GABA(A) receptor-associated protein, and synuclein-gamma). Pathway analysis identified oxidative phosphorylation, in particular, genes encoding complex 1 proteins that play a role in ubiquinone biosynthesis, to be the predominant gene set that was significantly regulated in a leptin-dependent manner. Thus, in addition to its effects on the expression of a broad range of neuropeptides, leptin may also exert more general influences on synaptic function in, and the bioenergetic state of, the PVN.

Leptin-regulated gene expression in MCF-7 breast cancer cells: mechanistic insights into leptin-regulated mammary tumor growth and progression

Obesity is a recently established risk factor for breast cancer incidence and mortality. A characteristic of obesity is elevated circulating levels of adipocyte-derived hormone leptin. Evidence indicates that leptin plays an important role in mammary tumor formation; however, the mechanisms involved are poorly understood. Toward better defining the role of leptin in breast cancer, we describe the identification of leptin-regulated genes in hormone-responsive Michigan Cancer Foundation-7 (MCF-7) human breast cancer cells using a microarray system. More than 64 leptin-regulated genes were identified including those for growth factors, cell cycle regulators, extracellular matrix (ECM) proteins, and genes associated with metastasis. Cell cycle genes up-regulated by leptin include cyclins D and G, cyclin-dependent kinase 2, p21, p27, and p16. Leptin suppressed the expression of transforming growth factor-beta , a cell cycle suppressor. Determining the significance of this effect, treatment of MCF-7 cells with TGFB1 abrogated leptin-stimulated proliferation. Leptin up-regulated the expression of connective tissue growth factor, villin 2, and basigin, factors that are associated with ECM and are known to impact tumor growth. Finally, leptin induced the expression of anti-apoptotic genes BCL2 and survivin, and reduced the expression of apoptotic genes. The effect of leptin on MCF-7 survival was evaluated via TUNEL assay and demonstrated a sixfold reduction in apoptosis in leptin-treated cells, compared with controls. These data suggest leptin promotes mammary tumor growth through multiple mechanisms, including regulating the cell cycle, apoptosis, and by modulating the extracellular environment. The identification of leptin-regulated genes begins to provide mechanistic links into the relationship between obesity and breast cancer incidence and morbidity.

Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line

Protein tyrosine phosphatase 1B (PTP1B) has recently been implicated in the regulation of body weight. A surprising phenotype of PTP1B-deficient mice is their resistance to diet-induced obesity. Since leptin is one of the primary hormones involved in the regulation of body weight and energy homeostasis, we investigated whether PTP1B affects leptin receptor (lepR) signaling directly. A mouse hypothalamic cell line, GT1-7, was established as a suitable cell model for the study of leptin signaling. Stimulation of GT1-7 cells by leptin caused tyrosine phosphorylation of endogenous STAT3 and activation of a STAT-dependent luciferase reporter gene. Over-expression of PTP1B in GT1-7 cells resulted in a dose-dependent decrease in endogenous JAK2 and STAT3 tyrosine phosphorylation compared with cells transfected with lepR alone. Consistent with inhibition of JAK–STAT signaling, PTP1B over-expression caused a dose-dependent decrease in leptin-induced, STAT-dependent luciferase reporter gene activation in GT1-7 cells. Furthermore, over-expression of PTP1B led to a decrease in mRNA accumulation of suppressor-of-cytokine-signalling-3 (SOCS3) and c- fos, genes that are acutely induced by leptin. Using gene microarray analysis, we confirmed that PTP1B reduces the level of gene expression of SOCS3 and showed that the expression level of other leptin-regulated genes was affected. Genes up-regulated by leptin were decreased in cells over-expressing PTP1B. Conversely, the expression of genes down-regulated by leptin was enhanced by PTP1B over-expression in GT1-7 cells. Our findings indicate that PTP1B is a negative regulator of leptin signaling and suggest that PTP1B inhibitors might be efficacious in the treatment of obesity by increasing leptin sensitivity.

Role for stearoyl-CoA desaturase-1 in leptin-mediated weight loss.

Leptin elicits a metabolic response that cannot be explained by its anorectic effects alone. To examine the mechanism underlying leptin's metabolic actions, we used transcription profiling to identify leptin-regulated genes in ob/ob liver. Leptin was found to specifically repress RNA levels and enzymatic activity of hepatic stearoyl-CoA desaturase-1 (SCD-1), which catalyzes the biosynthesis of monounsaturated fatty acids. Mice lacking SCD-1 were lean and hypermetabolic. ob/ob mice with mutations in SCD-1 were significantly less obese than ob/ob controls and had markedly increased energy expenditure. ob/ob mice with mutations in SCD-1 had histologically normal livers with significantly reduced triglyceride storage and VLDL (very low density lipoprotein) production. These findings suggest that down-regulation of SCD-1 is an important component of leptin's metabolic actions.

Effects of leptin deficiency and short-term repletion on hepatic gene expression in genetically obese mice.

By supplying most organs of the body with metabolic substrates, the liver plays a central role in maintaining energy balance. Hepatic metabolism of glucose, fatty acids, and lipoproteins is disrupted in the leptin-deficient obese (Lep(ob)/Lep(ob)) mouse, leading to hyperglycemia, steatosis, and hypercholesterolemia. Microarray expression profiles were used to identify transcriptional perturbations that underlie the altered hepatic physiology of Lep(ob)/Lep(ob) mice. A wide variety of genes involved in fatty acid metabolism are altered in expression, which suggests that both fatty acid synthesis and oxidation programs are activated in obese mice. The expression of a small subset of genes is upregulated by leptin deficiency, not modulated by caloric restriction, and markedly suppressed by short-term leptin treatment. Among these leptin-regulated genes, apolipoprotein A-IV is a strong candidate for mediating the atherogenic-resistant phenotype of Lep(ob)/Lep(ob) mice.

Leptin-specific patterns of gene expression in white adipose tissue

Leptin is a hormone that regulates body weight by decreasing food intake and increasing energy expenditure. ob/ob mice carry leptin mutations and are obese and hyperphagic. Leptin administration to lean and ob/ob mice activates a novel metabolic program that depletes adipose tissue. Although this response is physiologically distinct from that evident after food restriction, the molecular nature of these differences is as yet unknown. Expression monitoring of 6500 genes using oligonucleotide microarrays in wild-type, ob/ob, and transgenic mice expressing low levels of leptin revealed that differences in ambient leptin levels have dramatic effects on the phenotype of white adipose tissue. These data identified a large number of genes that are differentially expressed in ob/ob mice. To delineate the components of the transcriptional program specifically affected by leptin, the level of the same 6500 genes was monitored in wild-type and ob/ob mice at various times after leptin treatment or food restriction. A novel application of k-means clustering identified 8 clusters of adipose tissue genes whose expression was different between leptin treatment and food restriction in ob/ob mice and 10 such clusters in wild-type experiments. One of the clusters was repressed specifically by leptin in both wild-type and ob/ob mice and included several genes known to be regulated by SREBP-1/ADD1. Further studies confirmed that leptin decreases the levels of SREBP-1/ADD1 RNA and transcriptionally active SREBP-1/ADD1 protein in white adipose tissue. Future studies of the molecular basis for the apparent coordinate regulation of the other clusters of leptin-regulated genes may reveal additional mechanisms by which leptin exerts its weight-reducing effects.