Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.

Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.

Impact of the leptin receptor gene on pig performance and quality traits.

The recessive T allele of the missense polymorphism rs709596309 C > T of the leptin receptor gene is associated with intramuscular fat. However, its overall impact on pork production is still partial. In this work, we investigated the all-round effects of the TT genotype on lean growth efficiency and carcass, meat and fat quality using data from an experiment that compared the performance of 48 TT and 48 C- (24 CT and 24 CC) Duroc barrows. The TT pigs were less efficient for lean growth than the C- pigs. Although heavier, their carcasses had less lean content, were shorter and had lighter loins. Apart from increasing marbling and saturated fatty acid content, changes caused by the TT genotype in meat and fat quality are likely not enough to be perceived by consumers. The effect on visual marbling score exceeded that on intramuscular fat content, which suggests a direct influence of the T allele on the pattern of fat distribution in muscle. With current low-protein diets, the T allele is expected to be cost-effective only in niche markets where a very high level of marbling is critical.

Leptin signalling regulates transcriptional differences in granulosa cells from genetically obese mice but not the activation of NLRP3 inflammasome.

Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16 weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity.

Central leptin signaling deficiency induced by leptin receptor antagonist leads to hypothalamic proteomic remodeling.

AIMS: Leptin irresponsiveness, which is often associated with obesity, can have significant impacts on the hypothalamic proteome of individuals, including those who are lean. While mounting evidence on leptin irresponsiveness has focused on obese individuals, understanding the early molecular and proteomic changes associated with deficient hypothalamic leptin signaling in lean individuals is essential for early intervention and prevention of metabolic disorders. Leptin receptor antagonists block the binding of leptin to its receptors, potentially reducing its effects and used in cases where excessive leptin activity might be harmful. MATERIALS AND METHODS: In this work, we blocked the central actions of leptin in lean male adult Wistar rat by chronically administering intracerebroventricularly the superactive leptin receptor antagonist (SLA) (D23L/L39A/D40A/F41A) and investigated its impact on the hypothalamic proteome using label-free sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for quantitative proteomics. KEY FINDINGS: Our results show an accumulation of proteins involved in mRNA processing, mRNA stability, and translation in the hypothalamus of SLA-treated rats. Conversely, hypothalamic leptin signaling deficiency reduces the representation of proteins implicated in energy metabolism, neural circuitry, and neurotransmitter release. SIGNIFICANCE: The alterations in the adult rat hypothalamic proteome contribute to dysregulate appetite, metabolism, and energy balance, which are key factors in the development and progression of obesity and related metabolic disorders. Additionally, using bioinformatic analysis, we identified a series of transcription factors that are potentially involved in the upstream regulatory mechanisms responsible for the observed signature.

Central inhibition of HDAC6 re-sensitizes leptin signaling during obesity to induce profound weight loss.

Leptin resistance during excess weight gain significantly contributes to the recidivism of obesity to leptin-based pharmacological therapies. The mechanisms underlying the inhibition of leptin receptor (LepR) signaling during obesity are still elusive. Here, we report that histone deacetylase 6 (HDAC6) interacts with LepR, reducing the latter's activity, and that pharmacological inhibition of HDAC6 activity disrupts this interaction and augments leptin signaling. Treatment of diet-induced obese mice with blood-brain barrier (BBB)-permeable HDAC6 inhibitors profoundly reduces food intake and leads to potent weight loss without affecting the muscle mass. Genetic depletion of Hdac6 in Agouti-related protein (AgRP)-expressing neurons or administration with BBB-impermeable HDAC6 inhibitors results in a lack of such anti-obesity effect. Together, these findings represent the first report describing a mechanistically validated and pharmaceutically tractable therapeutic approach to directly increase LepR activity as well as identifying centrally but not peripherally acting HDAC6 inhibitors as potent leptin sensitizers and anti-obesity agents.

The Association Between the 5-Hydroxytryptamine Receptor 2A Gene Variants rs6311 and rs6313 and Obstructive Sleep Apnea in the Iranian Kurdish Population.

Introduction: Sleep is one of the most significant parts of everyone's life. Most people sleep for about one-third of their lives. Sleep disorders negatively impact the quality of life. Obstructive sleep apnea (OSA) is a severe sleep disorder that significantly impacts the patient's life and their family members. This study aimed to investigate the relationship between rs6313 and rs6311 polymorphisms in the serotonin receptor type 2A gene and OSA in the Kurdish population. Materials and Methods: The study's population comprises 100 OSA sufferers and 100 healthy people. Polysomnography diagnostic tests were done on both the patient and control groups. The polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) was used to investigate the relationship between OSA and LEPR gene polymorphisms. Results: Statistical analysis showed a significant relationship between genotype frequencies of patient and control groups of rs6311 with OSA in dominant [odds ratio (OR) = 5.203, p < 0.001) and codominant models (OR = 9.7, p < 0.001). Also, there was a significant relationship between genotype frequencies of patient and control groups of rs6313 with OSA in dominant (OR = 10.565, p < 0.001) and codominant models (OR = 5.938, p < 0.001). Conclusions: Findings from the study demonstrated that the two polymorphisms rs6311 and rs6313 could be effective at causing OSA; however, there was no correlation between the severity of the disease and either of the two polymorphisms.

Hydrogen sulfide regulates macrophage polarization and necroptosis to accelerate diabetic skin wound healing.

Hydrogen sulfide (H2S), recognized as the third gasotransmitter, plays a pivotal role in the pathophysiological processes of various diseases. Cystathionine γ-lyase (CSE) is the main enzyme for H2S production in the skin. However, effects and mechanisms of H2S in diabetic skin wound healing remain unclear. Our findings revealed a decrease in plasma H2S content in diabetic patients with skin wounds. CSE knockout (KO) diabetic mice resulted in delayed wound healing, reduced blood perfusion, and CD31 expression around the wounds. It also led to increased infiltration of inflammatory cells and M1-type macrophages, decreased collagen levels, α-smooth muscle actin (α-SMA), and proliferating cell nuclear antigen (PCNA) expression. Additionally, there were enhanced expressions of necroptosis related proteins, including receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like protein (MLKL). In comparison, sodium hydrosulfide (NaHS), H2S donor, accelerated skin wound healing in leptin receptor deficiency (db/db) mice. This acceleration was accompanied by increased blood perfusion and CD31 expression, reduced infiltration of inflammatory cells and M1-type macrophages, elevated collagen levels, α-SMA, and PCNA expressions, and decreased necroptosis-related protein expressions together with nuclear factor-κB (NF-κB) p65 phosphorylation. In conclusion, H2S regulates macrophage polarization and necroptosis, contributing to the acceleration of diabetic skin wound healing. These findings offer a novel strategy for the treatment of diabetic skin wounds.

Association of leptin receptor polymorphisms with susceptibility of non-small cell lung cancer: Evidence from 2249 subjects.

Non-small cell lung cancer (NSCLC) is increasing dramatically. It is believed that energy metabolism-related genes could play an important role in etiology of NSCLC. In this study, we sought to assess the correlation between three LEPR single nucleotide polymorphisms (rs1137101, rs1137100 and rs6588147) with NSCLS susceptibility. In total, 1193 NSCLC cases and 1056 controls were included. SNPscan™ genotyping method was used to analyze the genotypes of LEPR polymorphisms. Compared to rs6588147 GG in LEPR gene, this study identified a protective role of LEPR rs6588147 GA and GA/AA for the occurrence of NSCLC (GA vs. GG [p = 0.021] and GA/AA vs. GG [p = 0.030]). As well, we found that a protective role of LEPR rs6588147 for the occurrence of non-SCC subgroup (p < 0.05). By logistic regression analysis, we found that the rs6588147 A allele related genotypes might play a protective role for the occurrence of NSCLC in drinking, BMI ≥24 kg/m2, smoking and male subgroups. We also found that the rs1137101 A allele related genotypes played a protective role for the occurrence of NSCLC in male, younger participants (under 59 years) and overweight/obesity (BMI ≥24 kg/m2) subgroups. We found that LEPR Ars1037100Ars1037101Ars6588147 haplotype might play a protective role for the occurrence of NSCLC (p = 0.013). In addition, our findings indicated that LEPR rs1137100 G>A SNP might increase the risk of lymph node metastases (p = 0.038). This study highlights that LEPR rs6588147, rs1137101 genotypes and LEPR Ars1037100Ars1037101Ars6588147 haplotype are correlated with the occurrence of NSCLC. LEPR rs1137100 G>A SNP increases the risk of lymph node metastases.

Loss of GIPR in LEPR cells impairs glucose control by GIP and GIP:GLP-1 co-agonism without affecting body weight and food intake in mice.

OBJECTIVE: The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr). METHODS: Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice. RESULTS: Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the ß-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice. CONCLUSIONS: GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism.

Leptin Receptor Deficiency Impairs Lymph Node Development and Adaptive Immune Response.

Activation and clonal expansion of the Ag-specific adaptive immune response in the draining lymph node is essential to clearing influenza A virus infections. Activation sufficient for virus clearance is dependent on the lymph node's architectural organization that is maintained by stromal cells, chiefly fibroblastic reticular cells. During an analysis of influenza A virus clearance in leptin receptor knockout (DB/DB) mice, we observed that the DB/DB mice have markedly reduced numbers of lymph node fibroblastic reticular cells at the steady state. The reduction in lymph node fibroblastic reticular cells resulted in abnormal lymph node organization and diminished numbers of adaptive immune cells in the lymph nodes under homeostatic conditions. As a consequence, the DB/DB mice were impaired in their ability to generate an effective influenza-specific adaptive immune response, which prevented virus clearance. Using leptin receptor mutant mice with point mutations at distinct signaling sites in the leptin receptor, we were able to link the leptin receptor's signaling domain tyrosine 985, which does not contribute to obesity, to lymph node fibroblastic reticular cell development and function. These results demonstrate a novel role for leptin receptor signaling in regulating lymph node development in a manner that is crucial to the generation of Ag-specific adaptive immune responses.

Leptin and Leptin Receptor Polymorphisms in Infants and Their Parents: Correlation with Preterm Birth.

It has been proven that single-nucleotide polymorphisms (SNPs) in LEP and LEPR genes could predispose individuals to an increased risk of pregnancy adverse outcomes (PAOs) such as recurrent pregnancy loss (RPL) and pre-eclampsia. Preterm birth (PTB) is the leading cause of infant mortality. We decided to investigate the correlation between PTB and LEP and LEPR SNPs. The study cohort included families who underwent spontaneous PTB and control samples of families who had at-term-born (≥37 weeks of gestational age) children. Swabs were performed by rubbing the sticky end for about 30 s on the gum and on the inside of the cheek, allowing us to collect the flaking cells of the oral mucosa. Genotyping of the three SNPs-LEPRA668G, LEPG2548A and A19G-was carried out via an ARMS-MAMA real-time PCR procedure, as previously described. Regarding LEPG2548A, we found that the most expressed genotype in infants both in the preterm and the at-term group was AG; however, we did not discover any statistically significant difference (p = 0.97). Considering LEPA19G, none among the infants and parents were found to carry the AA genotype. No statistically significant differences were found between children, mothers and fathers belonging to preterm and at-term groups. We did not find a statistically significant association in newborns and their mother, but our results show a statistical correlation with the LEPRA668G genotype GG of the father. This fact can contribute to defining genetic risk factors for PTB. Further studies are certainly needed to better clarify the role of genetics in influencing preterm delivery.

The temporal and spatial pattern of leptin receptor-expressing cells in the developing mouse hypothalamus.

The arcuate nucleus is a crucial hypothalamic brain region involved in regulating body weight homeostasis. Neurons within the arcuate nucleus respond to peripheral metabolic signals, such as leptin, and relay these signals via neuronal projections to brain regions both within and outside the hypothalamus, ultimately causing changes in an animal's behaviour and physiology. There is a substantial amount of evidence to indicate that leptin is intimately involved with the postnatal development of arcuate nucleus melanocortin circuitry. Further, it is clear that leptin signalling directly in the arcuate nucleus is required for circuitry development. However, as leptin receptor long isoform (Leprb) mRNA is expressed in multiple nuclei within the developing hypothalamus, including the postsynaptic target regions of arcuate melanocortin projections, this raises the possibility that leptin also signals in these nuclei to promote circuitry development. Here, we used RT-qPCR and RNAscope® to reveal the spatio-temporal pattern of Leprb mRNA in the early postnatal mouse hypothalamus. We found that Leprb mRNA expression increased significantly in the arcuate nucleus, ventromedial nucleus and paraventricular nucleus of the hypothalamus from P8, in concert with the leptin surge. In the dorsomedial nucleus of the hypothalamus, increases in Leprb mRNA were slightly later, increasing significantly from P12. Using duplex RNAscope®, we found Leprb co-expressed with Sim1, Pou3f2, Mc4r and Bdnf in the paraventricular nucleus at P8. Together, these data suggest that leptin may signal in a subset of neurons postsynaptic to arcuate melanocortin neurons, as well as within the arcuate nucleus itself, to promote the formation of arcuate melanocortin circuitry during the early postnatal period.

Intergenerational inheritance induced by a high-fat diet causes hyperphagia and reduced hypothalamic sensitivity to insulin and leptin in the second-generation of rats.

OBJECTIVE: The aim was to investigate the intergenerational inheritance induced by a high-fat diet on sensitivity to insulin and leptin in the hypothalamic control of satiety in second-generation offspring, which were fed a control diet. METHODS: Progenitor rats were fed a high-fat or a control diet for 59 d until weaning. The first-generation and second-generation offspring were fed the control diet until 90 d of age. Body mass and adiposity index of the progenitors fed the high-fat diet and the second-generation offspring from progenitors fed the high-fat diet were evaluated as were the gene expression of DNA methyltransferase 3a, angiotensin-converting enzyme type 2, angiotensin II type 2 receptor, insulin and leptin signaling pathway (insulin receptor, leptin receptor, insulin receptor substrate 2, protein kinase B, signal transducer and transcriptional activator 3, pro-opiomelanocortin, and neuropeptide Agouti-related protein), superoxide dismutase activity, and the concentration of carbonyl protein and satiety-regulating neuropeptides, pro-opiomelanocortin and neuropeptide Agouti-related protein, in the hypothalamus. RESULTS: The progenitor group fed a high-fat diet showed increased insulin resistance and reduced insulin-secreting beta-cell function and reduced food intake, without changes in caloric intake. The second-generation offspring from progenitors fed a high-fat diet, compared with second-generation offspring from progenitors fed a control diet group, had decreased insulin-secreting beta-cell function and increased food and caloric intake, insulin resistance, body mass, and adiposity index. Furthermore, second-generation offspring from progenitors fed a high-fat diet had increased DNA methyltransferase 3a, neuropeptide Agouti-related protein, angiotensin II type 1 receptor, and nicotinamide adenine dinucleotide phosphate oxidase p47phox gene expression, superoxide dismutase activity, and neuropeptide Agouti-related protein concentration in the hypothalamus. In addition, there were reduced in gene expression of the insulin receptor, leptin receptor, insulin receptor substrate 2, pro-opiomelanocortin, angiotensin II type 2 receptor, angiotensin-converting enzyme type 2, and angiotensin-(1-7) receptor and pro-opiomelanocortin concentration in the second-generation offspring from progenitors fed the high-fat diet. CONCLUSIONS: Overall, progenitors fed a high-fat diet induced changes in the hypothalamic control of satiety of the second-generation offspring from progenitors fed the high-fat diet through intergenerational inheritance. These changes led to hyperphagia, alterations in the hypothalamic pathways of insulin, and leptin and adiposity index increase, favoring the occurrence of different cardiometabolic disorders in the second-generation offspring from progenitors fed the high-fat diet fed only with the control diet.

Obesity paradox in uveal melanoma: high body mass index is associated with low metastatic risk.

BACKGROUND: Metabolic factors and obesity may influence the development and progression of cancer. In this study, we examine their association with the risk of developing metastases of uveal melanoma. METHODS: Data on metabolic factors, medications, serum leptin levels, tumour leptin receptor RNA expression and clinical outcomes were examined in three cohorts. HRs for metastasis and cumulative incidences of melanoma-related mortality were calculated, and the levels of tumour leptin receptor expression were compared with prognostic factors including BAP1 mutation, and tumour cell morphology. RESULTS: Of 581 patients in the main cohort, 116 (20%) were obese and 7 (1 %) had metastatic disease at presentation. In univariate Cox regressions, tumour diameter, diabetes type II and use of insulin were associated with metastases, but patients with obesity had a lower risk. The beneficial prognostic implication of obesity was retained in multivariate regressions. In competing risk analyses, the incidence of melanoma-related mortality was significantly lower for patients with obesity. Serum leptin levels≥median were associated with a reduced risk for metastasis, independent of patient sex and cancer stage in a separate cohort (n=80). Similarly, in a third cohort (n=80), tumours with BAP1 mutation and epithelioid cells had higher leptin receptor RNA expression levels, which have a negative correlation with serum leptin levels. CONCLUSION: Obesity and elevated serum leptin levels are associated with a lower risk for developing metastases and dying from uveal melanoma.

Immunomodulatory leptin receptor+ sympathetic perineurial barrier cells protect against obesity by facilitating brown adipose tissue thermogenesis.

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor-positive (LepR+) sympathetic perineurial barrier cells (SPCs) present in mice and humans, which uniquely co-express Lepr and interleukin-33 (Il33) and ensheath AT sympathetic axon bundles. Brown ATs (BATs) of mice lacking IL-33 in SPCs (SPCΔIl33) had fewer regulatory T (Treg) cells and eosinophils, resulting in increased BAT inflammation. SPCΔIl33 mice were more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPCΔIl33 mice had impaired adaptive thermogenesis and were unresponsive to leptin-induced rescue of metabolic adaptation. We therefore identify LepR+ SPCs as a source of IL-33, which orchestrate an anti-inflammatory BAT environment, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR+IL-33+ SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research.

Leptin excites basolateral amygdala principal neurons and reduces food intake by LepRb-JAK2-PI3K-dependent depression of GIRK channels.

Leptin is an adipocyte-derived hormone that modulates food intake, energy balance, neuroendocrine status, thermogenesis, and cognition. Whereas a high density of leptin receptors has been detected in the basolateral amygdala (BLA) neurons, the physiological functions of leptin in the BLA have not been determined yet. We found that application of leptin excited BLA principal neurons by activation of the long form leptin receptor, LepRb. The LepRb-elicited excitation of BLA neurons was mediated by depression of the G protein-activated inwardly rectifying potassium (GIRK) channels. Janus Kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) were required for leptin-induced excitation of BLA neurons and depression of GIRK channels. Microinjection of leptin into the BLA reduced food intake via activation of LepRb, JAK2, and PI3K. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in vivo.

LRP1-deficient leptin receptor-positive cells in periodontal ligament tissue reduce alveolar bone mass by inhibiting bone formation.

OBJECTIVE: Leptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. DESIGN: Bone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. RESULTS: Bone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. CONCLUSION: LRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

Association of polymorphism in leptin receptor gene with susceptibility of adolescent idiopathic scoliosis.

PURPOSE: Abnormal leptin bioavailability has play key roles in the etiology of adolescent idiopathic scoliosis (AIS). Both leptin and its receptor levels may be modulated by the presence of genetic polymorphisms. This study aimed to evaluate the role of polymorphisms in the leptin (LEP) and its main receptor (LEPR) genes in the AIS susceptibility in girls. METHODS: A retrospective case-control study was conducted with 189 AIS and 240 controls. LEP rs2167270 and LEPR rs2767485 polymorphisms were genotyped using a TaqMan validated assay. Associations were evaluated by odds ratios (OR) and 95% confidence intervals (CI). RESULTS: The AIS group showed a predominance of girls under 18 years old (n = 140, 74.1%), 148 (78.3%) had low or normal BMI, 111 (58.7%) had Cobb ≥ 45º and 130 (68.7%) were skeletally mature. Minor allele frequencies of rs2167270 and rs2767485 were 35.7% and 18.3%, for AIS and 35.6% and 25.4% for controls, respectively. LEPR rs2767485 T and TC + TT were associated with higher risk of AIS (OR = 1.53; 95% CI = 1.09-2.13 and OR = 1.84; 95% CI = 1.69-2.01, respectively), since CC genotype was only present in the control group. In addition, the LEP rs2167270 GA + AA was more frequent in low weight group (BMI ≤ 24.9) of girls with AIS. There was no significant association between LEP rs2167270 and AIS susceptibility, and LEPR rs2767485 and BMI. CONCLUSION: The LEPR rs2767485 was associated with the genetic susceptibility of AIS and LEP rs2167270 with low BMI. These data can contribute to the identification of genetic biomarkers to improve the diagnosis and treatment.

CCK-sensitive C fibers activate NTS leptin receptor-expressing neurons via NMDA receptors.

The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR1. Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

ANGPTL4 binds to the leptin receptor to regulate ectopic bone formation.

Leptin protein was thought to be unique to leptin receptor (LepR), but the phenotypes of mice with mutation in LepR [db/db (diabetes)] and leptin [ob/ob (obese)] are not identical, and the cause remains unclear. Here, we show that db/db, but not ob/ob, mice had defect in tenotomy-induced heterotopic ossification (HO), implicating alternative ligand(s) for LepR might be involved. Ligand screening revealed that ANGPTL4 (angiopoietin-like protein 4), a stress and fasting-induced factor, was elicited from brown adipose tissue after tenotomy, bound to LepR on PRRX1+ mesenchymal cells at the HO site, thus promotes chondrogenesis and HO development. Disruption of LepR in PRRX1+ cells, or lineage ablation of LepR+ cells, or deletion of ANGPTL4 impeded chondrogenesis and HO in mice. Together, these findings identify ANGPTL4 as a ligand for LepR to regulate the formation of acquired HO.