Lepr Expression Research Articles

The Downregulation of the Liver Lipid Metabolism Induced by Hypothyroidism in Male Mice: Metabolic Flexibility Favors Compensatory Mechanisms in White Adipose Tissue

In mammals, the maintenance of energy homeostasis relies on complex mechanisms requiring tight synchronization between peripheral organs and the brain. Thyroid hormones (THs), through their pleiotropic actions, play a central role in these regulations. Hypothyroidism, which is characterized by low circulating TH levels, slows down the metabolism, which leads to a reduction in energy expenditure as well as in lipid and glucose metabolism. The objective of this study was to evaluate whether the metabolic deregulations induced by hypothyroidism could be avoided through regulatory mechanisms involved in metabolic flexibility. To this end, the response to induced hypothyroidism was compared in males from two mouse strains, the wild-derived WSB/EiJ mouse strain characterized by a diet-induced obesity (DIO) resistance due to its high metabolic flexibility phenotype and C57BL/6J mice, which are prone to DIO. The results show that propylthiouracil (PTU)-induced hypothyroidism led to metabolic deregulations, particularly a reduction in hepatic lipid synthesis in both strains. Furthermore, in contrast to the C57BL/6J mice, the WSB/EiJ mice were resistant to the metabolic dysregulations induced by hypothyroidism, mainly through enhanced lipid metabolism in their adipose tissue. Indeed, WSB/EiJ mice compensated for the decrease in hepatic lipid synthesis by mobilizing lipid reserves from white adipose tissue. Gene expression analysis revealed that hypothyroidism stimulated the hypothalamic orexigenic circuit in both strains, but there was unchanged melanocortin 4 receptor (Mc4r) and leptin receptor (LepR) expression in the hypothyroid WSB/EiJ mice strain, which reflects their adaptability to maintain their body weight, in contrast to C57BL/6J mice. Thus, this study showed that WSB/EiJ male mice displayed a resistance to the metabolic dysregulations induced by hypothyroidism through compensatory mechanisms. This highlights the importance of metabolic flexibility in the ability to adapt to disturbed circulating TH levels.

Regulatory role of Heparan sulfate in leptin signaling

Leptin, a hormone mainly secreted by adipocytes, has attracted significant attention since its discovery in 1994. Initially known for its role in appetite suppression and energy regulation, leptin is now recognized for its influence on various physiological processes, including immune response, bone formation, and reproduction. It exerts its effects by binding to receptors and initiating an intracellular signaling cascade. Heparan sulfate (HS) is known to regulate the intracellular signaling of various ligands. HS is present as the glycan portion of HSPGs on cell surfaces and in intercellular spaces, with diverse structures due to extensive sulfation and epimerization. Although HS chains on HSPGs are involved in many physiological processes, the detailed effects of HS chains on leptin signaling are not well understood.This study examined the role of HS chains on HSPGs in leptin signaling using Neuro2A cells expressing the full-length leptin receptor (LepR). We showed that cell surface HS was essential for efficient leptin signaling. Enzymatic degradation of HS significantly reduced leptin-induced phosphorylation of downstream molecules, such as signal transducer and activator of transcription 3 and p44/p42 Mitogen-activated protein kinase. In addition, HS regulated LepR expression and internalization, as treatment with HS-degrading enzymes decreased cell surface LepR. HS was also found to exhibit a weak interaction with LepR. Enzymatic removal of HS enhanced the interaction between LepR and low-density lipoprotein receptor-related protein 1, suggesting that HS negatively regulates this interaction. In conclusion, HS plays a significant role in modulating LepR availability on the cell surface, thereby influencing leptin signaling. These findings provide new insights into the complex regulation of leptin signaling and highlight potential therapeutic targets for metabolic disorders and obesity.

Reoxygenation improves reduced hypothalamic leptin responsiveness induced by intermittent hypoxia in obese rats

To evaluate the effects of intermittent hypoxia-reoxygenation (IHR) on body weight, diet and water intake, circulating metabolites, and responses to central leptin injection in a rat model of diet-induced obesity (DIO). Rat models of DIO established by 12-week high-fat diet (HFD) feeding were randomized into normoxia group (n=15), intermittent hypoxia group (6% O2, 30 cycles/h, 8 h/day for 4 weeks; n=15), and IHR group (2 weeks of intermittent hypoxia followed by 2 weeks of reoxygenation; n=15). Body weight, diet and water intake of the rats were recorded, and circulating leptin, IL-6, and Ang-II levels were detected. After IHR treatment, the rats received intracerebroventricular injection of 4 μg leptin, and the hypothalamus and liver were taken 1 h later for detecting POMC, FRA-1 and FRA-2 expressions in the hypothalamus using immunohistochemistry, POMC, pSTAT3 and LepR expressions in the hypothalamus using Western blotting, and LepR mRNA expression in the hypothalamus and liver using RT-PCR. The rats in intermittent hypoxia group showed significantly increased weight gain, food intake and elevated systemic inflammatory cytokine levels. Intermittent hypoxia obviously inhibited the expression of POMC, lowered the expressions of FRA-1 and pSTAT3, reduced the responsiveness of the rats to exogenous leptin, and downregulated the mRNA and protein expression of LepR. Two weeks of reoxygenation treatment obviously reduced intermittent hypoxia-induced weight gain and metabolic disorder and improved leptin sensitivity of the rats. Prolonged intermittent hypoxia impairs hypothalamic leptin signaling by downregulating LepR expression to promote weight gain in obese rats, which can be improved by reoxygenation treatment.

Abstract P147: Inappropriate Activation of the Leptin-Aldosterone-Endothelial Mineralocorticoid Receptor Axis Promotes Salt Sensitivity of Blood Pressure in Young Female Mice

While compelling clinical evidence indicates that women are more salt-sensitive than men, the mechanisms of the onset of women’s salt-sensitive blood pressure (SSBP) remain largely elusive. Herein, we employed salt-sensitive (SS)-Balb/C and salt-resistant (SR)-C57Bl/6 mice to investigate the etiopathology of SSBP in females. We hypothesized that a high-salt diet alters the leptin-aldosterone-endothelial mineralocorticoid receptor axis (ECMR), leading to SSBP and vascular dysfunction in young SS-female mice. Eleven-week-old female mice were fed a normal (0.4%, NSD) or a high salt diet (4%, HSD) for 7-days. We implanted radiotelemeters for BP monitoring at baseline, HSD, and in response to the leptin receptor (LepR) blockade combined with HSD. Following euthanasia, mesenteric arteries are isolated to measure vascular function via wire myography. HSD elevated mean arterial pressure in Balb/C (NSD: 98.4±2.3 vs. HSD: 105.1±0.9 mmHg) but not in C57 mice (NSD: 99.3±0.9 vs. HSD: 101.3±0.5 mmHg). However, SS-Balb/C mice showed superior natriuretic function in response to salt loading experiments (NSD: 13.3% vs. HSD: 42.3%) as opposed to C57 mice (NSD: 25.8% vs. HSD: 35.1%) (N=3). HSD feeding notably blunted endothelial relaxation to acetylcholine in Balb/c (NSD: 96.5% vs. HSD: 75.4%, N=7), an effect not seen in C57. SS-Balb/C mice failed to suppress aldosterone and adrenal aldosterone synthase (CYP11B2) levels (protein and transcript) compared to C57 mice. This concomitated a 1.8-fold increase in adrenal steroidogenic acute regulatory (StAR) mRNA in SS-Balb/C mice. HSD raised CYP11B2 protein levels in the visceral adipose tissue (VAT) of Balb/C mice. Leptin regulating CYP11B2 expression; we measured fat mass, leptin, and LepR expression. HSD enhanced fat accumulation (C57: +0.7% vs. Balb/c: +26.5%, N=12), leptin levels, and LepR transcript in the adrenal and VAT only in Balb/C mice. LepR blockade reduced BP in Balb/C mice fed a HSD (HSD: 105.1±0.9 vs. HSD+Allo-aca: 100.3±1.9 mmHg). HSD elevated ECMR expression only in Balb/C mice (N=6). To test the potential role of aldosterone-mediated ECMR activation in SSBP, we employed mice with selective overexpression of MR in endothelial cells (ECMR-OE). ECMR-OE young female mice exhibited impaired endothelial function (N=7), reproducing the effects of HSD. In conclusion, SSBP involves an over-activation of the leptin-aldosterone axis in both adrenal and adipose tissue, along with an escalated ECMR expression in females.

Adipokine receptor expression in mast cells is altered by specific ligands and proinflammatory cytokines.

Adipokines play essential roles in regulating a range of biological processes, but growing evidence indicates that they are also fundamental in immunological mechanisms and, primarily, inflammatory responses. Adipokines mediate their actions through specific receptors. However, although adipokine receptors are widely distributed in many cell and tissue types, limited data are available on their expression in mast cells (MCs) and, consequently, adipokine's significance in the modulation of MC activity within the tissues. In this study, we demonstrate that rat peritoneal MCs constitutively express the leptin receptor (i.e. LEPR), adiponectin receptors (i.e. ADIPOR1 and ADIPOR2) and the chemerin receptor (i.e. CMKLR1). We also found that LEPR, ADIPOR1, ADIPOR2 and CMKLR1 expression in MCs changes in response to stimulation by their specific ligands and some cytokines with potent proinflammatory properties. Furthermore, the involvement of intracellular signaling molecules in leptin-, adiponectin- and chemerin-induced MC response was analyzed. Overall, our findings suggest that adipokines leptin, adiponectin and chemerin can significantly affect the activity of MCs in various processes, especially during inflammation. These observations may contribute significantly to understanding the relationship between adipokines, immune mechanisms and diseases or conditions with an inflammatory component.

Characterization of adaptive expression regulation of yellowtail kingfish (Seriola lalandi) leptin, receptor, and receptor overlapping transcript genes in response to fasting and re-feeding strategies.

Leptins and other related genes have been proven to play vital roles in food intake, weight control, and other life activities. While the function of leptins in yellowtail kingfish (Seriola lalandi) has not yet been explored, in the present study, we investigated the structure and preliminary function of four leptin-related genes in S. lalandi. In detail, the sequence of two leptin genes (lepa and lepb), one leptin receptor gene (lepr), and one leptin receptor overlapping transcript (leprot) gene were obtained by homology cloning and RACE methods, in which lepa and lepb have similar structure. Moreover, homologous sequence alignment and evolutionary analysis of all four genes were clustered with Seriola dumerili. The tissue distribution of these four genes in thirteen tissues of yellowtail kingfish was detected by RT-qPCR. Both lepa and leprot were highly expressed in the brain and ovary, while lepb was highly expressed in the pituitary, gill, muscle, and ovary; lepr was highly expressed in the gill, kidney, and ovary. Additionally, these four genes also played roles in embryo development and early growth and development of larvae and juveniles of yellowtail kingfish. Finally, the function of leptin and leptin-related genes was investigated during fasting and re-feeding adaption of yellowtail kingfish. The results showed that these four genes have different regulation functions in five tissues; for example, the mRNA levels of lepa, lepr, and leprot in the brain decreased during fasting and immediately increased after re-feeding, while the mRNA level of lepb did not show significant fluctuation during starvation but significantly lowered after re-feeding. However, lepa and lepb mRNA levels were significantly elevated during fasting and returned to control levels after re-feeding, and there were no significant changes in the expression of lepr and leprot in the liver during fasting and after re-feeding. Moreover, the body mass of fish in the experimental group was measured, and compensatory growth was found after the resumption of feeding. These results suggested that leptin and receptor genes play different functions in different tissues to regulate the physiological state of fish in food deficiency and gain processes.

High Salt Diet Induces Aldosterone Synthase Elevation in Adrenal and Visceral Adipose Tissue - A Potential Culprit for Unsuppressed Aldosterone Levels in Female BALB/c Mice

Women are more salt-sensitive than men, irrespective of age, menopausal status, or ethnic background. Nonetheless, the mechanisms predisposing women to salt sensitivity of blood pressure (SSBP) are poorly understood, notably due to the diffculty in finding appropriate animal models. Previously, we demonstrated that female BALB/c mice spontaneously develop SSBP, likely through an inability to reduce the expression of adrenal aldosterone synthase (CYP11B2) appropriately, which leads to unsuppressed aldosterone levels in response to high salt intake. Herein, we aim to decipher the mechanism involved in SSBP in female BALB/c mice by comparing them to female salt-resistant C57BL/6 mice. Eleven-week-old female BALB/c and C57BL/6J mice were fed either a normal (0.4%, NSD) or a high salt diet (4%, HSD) for seven days. HSD significantly reduced adrenal aldosterone synthase transcript and protein expression in female C57BL/6 mice compared to those on a NSD. Conversely, HSD substantially increased CYP11B2 levels in BALB/c female mice when compared to strain-matched NSD and C57BL/6 NSD mice (2-way ANOVA, p<0.05). HSD induced a more significant aldosterone drop in C57BL/6 versus BALB/c mice (C57BL/6: 72% vs. BALB/c: 49%). Remarkably, HSD led to an increase in CYP11B2 in the visceral adipose tissue (VAT) of BALB/c mice in comparison to strain-matched NSD mice, while no such alteration was observed between C57BL/6 NSD and HSD. We previously showed that leptin regulates CYP11B2 expression. We report here that HSD increased leptin receptor (lepR) expression in adrenal and VAT from BALB/c mice in comparison to both strain-matched NSD and C57BL/6 NSD. Baseline levels of leptin were similar in C57BL/6 and BALB/c mice. However, BALB/c mice on HSD displayed increased levels of leptin compared to its strain-matched NSD mice, while no such alteration was observed in C57BL/6 mice. Furthermore, female BALB/c mice on HSD displayed elevated endothelial cell-specific mineralocorticoid receptor (ECMR) expression compared to female C57BL/6 NSD and HSD mice (2-way ANOVA, p<0.05). Endothelium-dependent relaxation to acetylcholine was significantly reduced in the female BALB/c mice on HSD compared to strain-matched NSD and C57BL/6 NSD (n = 3, 2-way ANOVA, P < 0.05). Interestingly, on NSD, C57BL/6 mice present a better endothelial function than BALB/c, which was not altered by HSD. Collectively, our data supports an overactivation of the leptin-CYP11B2-aldosterone axis in both adrenals and VAT, leading to inappropriate aldosterone levels, which, associated with higher ECMR expression, induce endothelial dysfunction in BALB/c mice. Together, these mechanisms are likely the cause of SSBP in female BALB/c mice. Funding: R01HL130301, R01HL155265 (E.J.B.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Leptin receptor reactivation restores brain function in early-life Lepr-deficient mice.

Obesity is a chronic disease caused by excessive fat accumulation that impacts the body and brain health. Insufficient leptin or leptin receptor (LepR) is involved in the disease pathogenesis. Leptin is involved with several neurological processes, and it has crucial developmental roles. We have previously demonstrated that leptin deficiency in early life leads to permanent developmental problems in young adult mice, including an imbalance in energy homeostasis, alterations in melanocortin and the reproductive system and a reduction in brain mass. Given that in humans, obesity has been associated with brain atrophy and cognitive impairment, it is important to determine the long-term consequences of early-life leptin deficiency on brain structure and memory function. Here, we demonstrate that leptin-deficient (LepOb) mice exhibit altered brain volume, decreased neurogenesis and memory impairment. Similar effects were observed in animals that do not express the LepR (LepRNull). Interestingly, restoring the expression of LepR in 10-week-old mice reverses brain atrophy, in addition to neurogenesis and memory impairments in older animals. Our findings indicate that leptin deficiency impairs brain development and memory, which are reversible by restoring leptin signalling in adulthood.

Triphenyltin induced darker body coloration by disrupting melanocortin system and pteridine metabolic pathway in a reef fish, Amphiprion ocellaris

Triphenyltin (TPT) is a typical persistent organic pollutant whose occurrence in coral reef ecosystems may threaten the survival of reef fishes. In this study, a brightly colored representative reef fish, Amphiprion ocellaris was used to explore the effects of TPT at environmental levels (1, 10, and 100 ng/L) on skin pigment synthesis. After the fish were exposed to TPT for 60 days, the skin became darker, owing to an increase in the relative area of black stripes, a decrease in orange color values while an increase in brown color values, and an increase in the number of melanocytes in the orange part of the skin tissues. To explore the mechanisms by which TPT induces darker body coloration, the enzymatic activity and gene expression levels of the members of melanocortin system that affect melanin synthesis were evaluated. Leptin levels and lepr expression were found to be increased after TPT exposure, which likely contributed to the increase found in pomc expression and α-melanocyte-stimulating hormone (α-MSH) levels. Then Tyr activity and mc1r, tyr, tyrp1, mitf, and dct were upregulated, ultimately increasing melanin levels. Importantly, RT-qPCR results were consistent with the transcriptome analysis of trends in lepr and pomc expression. Because the orange color values decreased, pterin levels and the pteridine metabolic pathway were also evaluated. The results showed that TPT induced BH4 levels and spr, xdh, and gch1 expression associated with pteridine synthesis decreased, ultimately decreasing the colored pterin content (sepiapterin). We conclude that TPT exposure interferes with the melanocortin system and pteridine metabolic pathway to increase melanin and decrease colored pterin levels, leading to darker body coloration in A. ocellaris. Given the importance of body coloration for the survival and reproduction of reef fishes, studies on the effects of pollutants (others alongside TPT) on body coloration are of high priority.

Phenotypic and genetic analysis of sexual dimorphism in growth, feed intake and feed conversion efficiency in yellow catfish

Sexual dimorphism in growth has been frequently observed in fish species, which encourages the production of mono-sex population in aquaculture. Besides growth, feed intake and feed conversion efficiency (FCE) are two key traits for sustainable aquaculture. In this study, yellow catfish (Pelteobagrus fulvidraco), an important aquaculture fish with significantly sexual size dimorphism, was used as a model to investigate the associations among phenotypic traits, candidate SNPs and correlated gene expression. Sexual dimorphism in body length and body weight were observed as early as 68 days post hatching. One SNP in corticotropin releasing hormone receptor 1 (crhr1) has been shown to be significantly associated with body length and body weight but slightly differ between males and females. Compared to female fish, male fish had higher levels of feed intake and higher FCE, as well as higher expression of stat5b and agrp in liver and hypothalamus, and lower expression of crhr1, pomca and lepr in pituitary and hypothalamus. The high expression levels of stat5b and agrp as well as low level of crhr1 were positively associated with high FCE in both sexes, whereas the higher expressions of pomca and lepr were negatively with FCE only in female fish. Finally, we showed that the identified SNPs in stat5b and crhr1 were significantly associated with growth and FCE in yellow catfish but slightly differ between males and females. These results suggest that sexual dimorphism in phenotypic traits might be caused by sex difference in genetic architecture and gene regulation. Our study not only suggest that feed intake and feed efficiency are associated with the sexual size dimorphism, but also identify potential genes and genomic markers associated with these important traits that would benefit the genetic breeding in yellow catfish.

Pilot Study of the DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 Gene Expression in Peripheral Blood Leukocytes in Schizophrenic Patients with Metabolic Syndrome

—Many individuals with schizophrenia also suffer from metabolic syndrome (MetS), which is a major risk factor for the development of cardiovascular disroders associated with a heavy burden of disease, as well as with premature death of patients. This study investigated the expression of 7 genes potentially important for the development of metabolic syndrome. QuantiGene Plex 2.0 technology was used to measure how 7 studied genes (DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 (MDR1)) were expressed in leukocytes in 60 recently admitted patients with schizophrenia who had been on treatment with antipsychotic drugs. The preliminary results of our study show a change in the expression of the FTO gene in schizophrenic males with metabolic disorders, however, further studies are needed to determine the role of disturbances in the expression of this gene in the development of the metabolic syndrome in patients with schizophrenia.

Suppression of food intake by Glp1r/Lepr-coexpressing neurons prevents obesity in mouse models.

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A potentially unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-Seq of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance caused by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.

Red and golden tomato administration improves fat diet-induced hepatic steatosis in rats by modulating HNF4α, Lepr, and GK expression.

Nonalcoholic fatty liver disease (NAFLD), characterized by lipid accumulation within hepatocytes exceeding 5% of liver weight, is strongly related to metabolic disorders, obesity, and diabetes and represents a health emergency worldwide. There is no standard therapy available for NAFLD. Lifestyle intervention, including phytonutrient intake, is key in preventing NAFLD development and progression. We used a rat model of NAFLD to evaluate the effect of dietary supplementation with red tomato (RT) and golden tomato (GT)-a patented mix of fruit with varying degrees of ripeness and particularly rich in naringenin and chlorogenic acid-after steatosis development. We assessed the effects on body weight, metabolic profile, and hepatic steatosis. We found a correlation between the amelioration of all the parameters and the liver gene expression. Our results showed that, together with the reversion of steatosis, the consumption of RT and GT can cause a significant reduction in triglycerides, low-density lipoprotein-cholesterol, fasting glucose, and homeostasis model assessment index. Meanwhile, we observed an increase in high-density lipoprotein-cholesterol according to the amelioration of the general lipidic profile. Regarding hepatic gene expression, we found the upregulation of Gk and Hnf4α involved in metabolic homeostasis, Lepr involved in adipokine signaling, and Il6 and Tnf involved in inflammatory response. Taken together, our results suggest that dietary intake of red and golden tomatoes, as a nutraceutical approach, has potential in preventing and therapeutics of NAFLD.

Bifidobacterium animalis subspecies lactis HN019 can reduce the sequelae of experimental periodontitis in rats modulating intestinal parameters, expression of lipogenic genes, and levels of hepatic steatosis.

To determine whether Bifidobacterium animalis subspecies lactis HN019 (B. lactis HN019) can reduce the sequelae of experimental periodontitis (EP) in rats modulating systemic parameters. This study evaluated the effects of probiotic therapy (PROB) in the prevention of local and systemic damage resulting from EP. Forty-eight rats were allocated into four groups: C (control), PROB, EP, and EP-PROB. PROB (1 × 1010 CFU/mL) administration lasted 8 weeks and PE was induced on the 7th week by placing ligature on the animals' lower first molars. All animals were euthanized in the 9th week of the experiment. Biomolecular analyses, RT-PCR, and histomorphometric analyses were performed. The data obtained were analyzed statistically (ANOVA, Tukey, p < .05). The EP group had higher dyslipidemia when compared to the C group, as well as higher levels of insulin resistance, proteinuria levels, percentages of systolic blood pressure, percentage of fatty hepatocytes in the liver, and expression of adipokines was up-regulated (LEPR, NAMPT, and FABP4). All these parameters (except insulin resistance, systolic blood pressure, LEPR and FABP4 gene expression) were reduced in the EP-PROB group when compared to the EP group. The EP group had lower villus height and crypt depth, as well as a greater reduction in Bacteroidetes and a greater increase in Firmicutes when compared to the EP-PROB group. Greater alveolar bone loss was observed in the EP group when compared to the EP-PROB group. Bifidobacterium lactis HN019 can reduce the sequelae of EP in rats modulating intestinal parameters, attenuating expression of lipogenic genes and hepatic steatosis.

P-796 Single-cell transcriptomics unveils a role for leptin receptor in human endometrial mesenchymal stromal/stem cells

Abstract Study question What are the characteristics of leptin receptor (LepR) expressing cells within the endometrial mesenchymal stromal/stem cells (eMSCs) population? Summary answer LepR+ eMSCs display better stem-cell capacities as well as distinctively quiescent properties compared to eMSCs and LepR- eMSCs. What is known already Single-cell RNA sequencing (scRNA-seq) reveals that cultured eMSCs are heterogeneous. LepR is highly expressed in bone marrow MSCs, and LepR+ cells contribute towards the formation of fibroblast colony-forming units (CFU-F). Mouse LepR+ MSCs are quiescent and activate upon injuries to form osteoblasts and adipocytes in the bone marrow. In this study, we hypothesized that LepR+ cells play a critical role in the stem cell population in human endometrium. Study design, size, duration We performed scRNA-seq on magnetic bead selected eMSCs (CD140b+CD146+ cells). Functional stem cell assays such as clonogenicity and serial subcloning were performed on the different stromal subsets (eMSC, LepR- eMSC, and LepR+ eMSC) isolated by fluorescence-activated cell sorting (FACS) cultured endometrial stromal cells. The cell cycle analysis was evaluated by multicolor flow cytometry on freshly isolated endometrial stromal cells. Participants/materials, setting, methods The endometrial samples were collected from women aged 37–53 years with regular menstrual cycles undergoing hysterectomy. The expression and localization of LepR in human endometrium were determined by flow cytometry and immunohistochemistry, respectively. The in vitro colony-forming and self-renewal abilities were assessed on FACS sorted endometrial stromal populations. The cell cycle status of the stromal subsets was determined by flow cytometry and the quiescent property was determined by qPCR (RB1, RBL2, CDKN1A, CDKN1B, and E2F4). Main results and the role of chance Clustering analysis of the scRNA-seq data revealed 5 subpopulations (SP0-4) of cultured eMSCs. A high proportion (&amp;gt;90%) of the cells in SP3 and SP4 were at the G1 phase. The majority of the SP3 cells (92%) were located at the root of the cell trajectory and pseudo time inference analysis. The expression of LepR was highly specific in SP3 suggesting LepR eMSCs are potentially the origin progenitors.The expression of LepR in endometrial stromal cells was 2.61 ± 1.43%, and in eMSCs was 25.59 ± 19.04%. In vitro experiments demonstrated that LepR+ eMSCs exhibit better stem-cell properties, including clonogenic and self-renewal ability, than its negative counterparts. QPCR validation of cell-quiescence associated genes revealed higher expression of RB1, RBL2, and CDKN1B in the LepR+ eMSCs than eMSCs and LepR- eMSCs. In addition, multicolor flow analysis on freshly isolated endometrial cells revealed significantly higher proportion of LepR+ eMSCs are in the G0 phase of the cell cycle when compared to eMSCs and LepR- eMSCs. The above results indicate in vivo LepR+ eMSCs are relatively more quiescent. Limitations, reasons for caution The mechanism involved in maintaining or activating quiescent LepR+ eMSCs remains unknown. The role of LepR+ is likely to be different in human compared to mouse. We also lack an in vivo model for studying LepR+ eMSCs during endometrial repair. Wider implications of the findings Our findings extend the understanding of heterogeneous human endometrial mesenchymal stem cells and unveil a new role for LepR in endometrial regeneration. Trial registration number not applicatble

1781-P: Leptin Regulates Delta-Cell Somatostatin Secretion from Human and Mouse Islets

The delta cell is uniquely positioned in the islet to integrate local signals and circulating nutrient cues to regulate alpha and beta cell function through somatostatin (SST) secretion. In type 2 diabetes (T2D), delta cell responses to ambient glucose are disproportionate and consequently alter insulin and glucagon secretion leading to dysglycemia. However, we know relatively little about the mechanisms influencing delta cells. Thus, understanding factors that regulate delta cell SST secretion may reveal pathogenic mechanisms contributing to T2D and direct new therapies to achieve glucose homeostasis. Leptin is a circulating hormone that potently inhibits insulin and glucagon secretion, but notably, the leptin receptor (LepR) is exclusively expressed on delta cells of human islets. Our preliminary studies demonstrate for the first time that leptin stimulates SST secretion from human islets. Leptin-induced SST corresponded with decreased glucagon and insulin secretion. Leptin effects were conserved in mouse islets and required delta cell specific LepR expression. Our single-cell RNA-seq studies revealed Stat3 target genes become depleted in delta cells that lack LepR, suggesting canonical leptin signals regulate transcriptional activity in delta cells. Along these lines, delta cell specific Stat3 KO islets (Sst-Cre+/wt;Stat3fl/fl) do not respond to leptin. On-going studies in human islets employ pharmacologic and genetic interventions with live functional imaging and unbiased approaches that will reveal the mechanisms of leptin action in human delta cells. These studies are the first to describe leptin effects on delta cells and uncover a unifying mechanism whereby leptin acts indirectly on alpha and beta cells through paracrine SST signaling. As such, our findings are expected to reveal new signaling mechanisms governing islet function that potentially exert therapeutic benefits for T2D. Disclosure S.M.Hartig: None. A.Cox: None.

Influence of Leptin on the Secretion of Growth Hormone in Ewes under Different Photoperiodic Conditions.

Leptin is an adipokine with a pleiotropic impact on many physiological processes, including hypothalamic-pituitary-somatotropic (HPS) axis activity, which plays a key role in regulating mammalian metabolism. Leptin insensitivity/resistance is a pathological condition in humans, but in seasonal animals, it is a physiological adaptation. Therefore, these animals represent a promising model for studying this phenomenon. This study aimed to determine the influence of leptin on the activity of the HPS axis. Two in vivo experiments performed during short- and long-day photoperiods were conducted on 12 ewes per experiment, and the ewes were divided randomly into 2 groups. The arcuate nucleus, paraventricular nucleus, anterior pituitary (AP) tissues, and blood were collected. The concentration of growth hormone (GH) was measured in the blood, and the relative expression of GHRH, SST, GHRHR, SSTR1, SSTR2, SSTR3, SSTR5, LEPR, and GH was measured in the collected brain structures. The study showed that the photoperiod, and therefore leptin sensitivity, plays an important role in regulating HPS axis activity in the seasonal ewe. However, leptin influences the release of GH in a season-dependent manner, and its effect seems to be targeted at the posttranscriptional stages of GH secretion.

Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells

The lineage of periodontal ligament (PDL) stem cells contributes to alveolar bone (AB) and cementum formation, which are essential for tooth-jawbone attachment. Leptin receptor (LepR), a skeletal stem cell marker, is expressed in PDL; however, the stem cell capacity of LepR+ PDL cells remains unclear. We used a Cre/LoxP-based approach and detected LepR-cre-labeled cells in the perivascular around the root apex; their number increased with age. In the juvenile stage, LepR+ PDL cells differentiated into AB-embedded osteocytes rather than cementocytes, but their contribution to both increased with age. The frequency of LepR+ PDL cell-derived lineages in hard tissue was < 20% per total cells at 1-year-old. Similarly, LepR+ PDL cells differentiated into osteocytes following tooth extraction, but their frequency was < 9%. Additionally, both LepR+ and LepR− PDL cells demonstrated spheroid-forming capacity, which is an indicator of self-renewal. These results indicate that both LepR+ and LepR− PDL populations contributed to hard tissue formation. LepR− PDL cells increased the expression of LepR during spheroid formation, suggesting that the LepR− PDL cells may hierarchically sit upstream of LepR+ PDL cells. Collectively, the origin of hard tissue-forming cells in the PDL is heterogeneous, some of which express LepR.

Reproductive phase-dependent and sexually dimorphic expression of leptin and its receptor in different parts of brain of spotted snakehead Channa punctata.

The reproductive phase-wise leptin (lep) and its receptor (lepr) expression in different parts of the brain of adult male and female spotted snakehead Channa punctata reveals sexual dimorphism in the brain leptin system. In anterior, middle and posterior parts of the brain of males, a maximum lep was observed in resting, spawning and postspawning reproductive phases, respectively. In females, a high level of lep was seen during the preparatory phase in the anterior brain, preparatory and postspawning phases in the middle brain and resting and postspawning phases in the posterior brain. Nonetheless, the transcript level of lepr was recorded highest during the spawning phase, irrespective of sex and region of the brain. Regardless of the reproductive state of fishes, lep and lepr were seen considerably high in middle and posterior parts of male brain than that of female, implying the involvement of factors other than sex steroids for sex-related variation in the leptin system in these regions of the brain. Nonetheless, no sex difference was evidenced in the expression of either ligand or its receptor in the anterior brain. In summary, the presence of lep and lepr in different regions of the brain and variation in their expression depending on sex and reproductive phases raise the possibility of pivotal actions of leptin in influencing neuronal circuitry and thereby reproductive functions.

Infusion of Cultured Endothelial Progenitor Cells Boosts Hematopoietic Recovery and Increases the Expression of LepR+ Cells after Irradiation

The hemopoietic system's function of producing blood cells and immune cells during thorough life relies on the Maintainance and differentiation of hematopoietic stem cells (HSCs). HSCs activities were tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow. However, during the routine treatment of hematopoietic diseases, such as chemotherapy and radiotherapy, the niche was inevitably impaired which is one of the essential causes of hematopoietic suppression. Our previous research results showed that infusion of cultured endothelial progenitor cells (EPCs) could boost hematopoietic recovery after HSCT. And the latent mechanisms could be that EPCs can differentiate into endothelial cells (ECs) in vivo. However, the roles of EPCs play in the bone marrow niche and the specific mechanism remain unknown. Here we designed further to investigate the effect and mechanism of EPCs in boosting bone marrow microenvironment recovery after total body irradiation (TBI). This may offer a new cue for therapy of hematopoietic suppression after the treatment of malignant hematological diseases. Bone marrow-derived Primary EPCs isolation and culture were described as before. Briefly, Bone marrow cells from Balb/c or C57BL/6J mice aged 6-8 weeks were cultured with EGM-2 medium which was changed in 36 h, the adherent cells formed colonies identified as elongated sprouting cells radiating from a central core of round cells and identified as VEGFR2+; CD133+; CD144+; SCA1+ by FACS on days 4-6. To assess the effects of the EPCs in boosting hematopoiesis, Balb/c mice underwent TBI with a lethal dose and were injected withEPCs or the same volume of PBS via tail vein in 4-6 hours. The survival of the EPC group was elevated ( survival: 8/9 vs 0/13; p＜0.001). C57BL/6J mice underwent TBI with a sublethal dose and were injected with the cultured EPCs isolated from C57BL/6-Tg(CAG-EGFP) mice or the same volume of PBS via tail vein in 4-6 hours. Results showed that EPCs boost the recovery of hematopoietic cells in the bone marrow and peripheral blood cells; Immunofluence (IF) staining showed that Infused EPCs with GFP fluorescence were home to bone marrow and differentiated into ECs which were marked with endomucin (EMCN); Moreover, assessments of the alteration of bone marrow niche using H&E staining revealed that infusion of EPCs reduced the accumulation of fat and using multi-analyte flow assay showed that mice infused with EPCs express a higher level of M-CSF and TGF-β in plasma while the level of SCF and IL-6 both in plasma and bone marrow supernatant were lower compared to TBI. Furthermore, to assess the change of sinusoidal niche, we used IF staining and found that the sinusoids were dilated and perisinusoidal stromal cells identified as LepR+ were massively lost on the 7th day after TBI while the dilated sinusoidal lumens were narrowed (11.05±1.789 vs 20.86±4.573，p=0.0258) and LepR+ cells were upregulated on the 21st day after the infusion of EPCs which indicated that the sinusoidal niche has a better recovery compared to TBI. In Conclusion, EPCs could differentiate into endothelial cells in vivo boosting the recovery of the hematopoietic system. And the potential mechanism is that EPCs repair the bone marrow sinusoidal niche damaged by irradiation.