High Doses Of Leptin Research Articles

(092) Leptin Increases T-cells in Testis to Modulate Leydig Stem Cell Function

Abstract Introduction Testis microenvironment which is comprised of cells like Sertoli and peritubular cells releases paracrine factors which could potentially modulate testicular functions. In our previous studies, we identified Leptin as a key paracrine factor in the testicular microenvironment, released by Sertoli and peritubular cells and goes to bind its receptor in Leydig cells. Once this association happens it further induces the expression of the desert hedgehog signaling pathway, which is known to play an important role in the Leydig Stem cell’s differentiation to Adult Lydig Cells, which are capable of producing testosterone. However, one of the limitations in our study was that the model we used were from human cells that were extracted from testes’ biopsies which lacked an intact immune microenvironment. In the present study we explored the influence of Leptin on an intact testicular immune microenvironment. Objective To explore the influence of Leptin on an intact testicular immune microenvironment. Methods We used 6-weeks old C57BL6 mice which were exposed to two different concentrations of Leptin, 10 and 100 mg/ml for seven days. After seven days of treatment, these animals were humanly sacrificed and we collected their blood and testes. Blood was subjected to the CBC differential profile performed using EDTA anti-coagulated blood samples with a Hemavet Mascot Multispecies Hematology System Counter 1500R (CDC Technologies, Inc., Oxford, CT). Samples were counted no longer than five minutes after blood was drawn. In addition to that, the testes were used and subjected to comprehensive immunophenotyping that allowed us to evaluate in detail the impact of Leptin treatment at different doses on different immune cell types which are found within the testes. Results Results showed that Leptin at low dose (10mg/ml) showed increased levels of serum Testosterone (13.20 + 0.37 ng/dl) but were not significant, LH and FSH (LH 0.016 + 0.02 ng/ml, FSH in 51.71 + 12.93 ng/ml) showed significant (p<0.05) increase. On the contrary, animals which received Leptin at 100mg/ml showed no significant changes in hormone levels. CBC profiling suggested that amongst all the factors that were compared, Lymphocytes and Platelets were significantly influenced specifically upon low doses of Leptin. Interestingly, comprehensive immunophenotyping highlighted that the animals that received Leptin at low doses had a significant increase in the total number of CD4+ (helper T cells) and CD8+ (Cytotoxic T lymphocytes) cells. This pattern was not observed in animals which received high doses of Leptin. Conclusions The observation made in this study highlights unexplored aspects of influence of paracrine factors released by Sertoli and peritubular myoid cells on modulating immune microenvironment within the testis to potentially influence testosterone production. In future studies we will be exploring how the changes in the different immune cell types are correlated directly with the increase in T cells and how this correlation can influence the Leydig Stem Cell differentiation into Leydig Adult Cells. Disclosure No

Restlessness and an Increased Urge to Move (Drive for Activity) in Anorexia Nervosa May Strengthen Personal Motivation to Maintain Caloric Restriction and May Augment Body Awareness and Proprioception: A Lesson From Leptin Administration in Anorexia Nervosa.

Anorexia nervosa (AN), a disorder of voluntary food restriction leading to severe weight loss in female adolescents, remains an enigma. In particular, the appropriation of the starved thin body into the self-concept in AN is a process insufficiently researched and still poorly understood. Healthy humans undergoing starvation experience a slowing of movements and avoid voluntary exercise. By contrast, AN tends to be not infrequently associated with voluntary, sometimes excessive and/or compulsive exercise. Such deliberate exercise, not reported in starvation, seems to be facilitated by an increased urge for movement and physical restlessness, particular to AN. The increased urge to move would reflect spontaneous daily activity, the energy expended for everything that is not sleeping, eating, or voluntary exercise. Our hypothesis is that the starvation-induced increased urge to move and restlessness may promote the development of AN. Reversal of the fasting state, by either high caloric food or by leptin administration, would be expected to reduce restlessness and the increased urge to move along with improvement in other symptoms in AN. This review explores the idea that such restless activation in AN, in itself and through accelerating body weight loss, might foster the integration of the starving body into the self-concept by (1) enhancing the person’s sense of self-control and sense of achievement and (2) through invigorating proprioception and through intensifying the perception of the changing body shape. (3) Tentative evidence from studies piloting leptin administration in chronic AN patients which support this hypothesis is reviewed. The findings show that short term administration of high doses of leptin indeed mitigated depressive feelings, inner tension, intrusive thoughts of food, and the increased urge to be physically active, easing the way to recovery, yet had little influence on the patients’ personal commitment to remain at a low weight. Full recovery then requires resolution of the individuals’ personal unresolved psychological conflicts through psychotherapy and frequently needs specialized treatment approaches to address psychiatric co-morbidities. AN might be conceptualized as a hereditary form of starvation resistance, facilitated by the effects of starvation on fitness allowing for an exceptionally intense personal commitment to perpetuate food restriction.

Local administration of low doses of exogenous BMP2 and leptin promotes ectopic bone regeneration in leptin-deficient mice.

Obesity and leptin deficiency are associated with compromised bone regeneration. This study aims to investigate the role of locally administrated low-dose BMP2+leptin on bone regeneration in leptin-deficient obese (ob/ob) mice. Wildtype (WT) and ob/ob mice were divided into 3 groups (4mice/group): BMP2 (5μg) group, BMP2+low-dose leptin (1μg) group, and BMP2+high-dose leptin (2.5μg) group. WT mice were used as control mice. An equal size absorbable collagen sponge was prepared by loading the BMP2 or/and leptin and implanted subcutaneously. After 19 days, samples were collected and analyzed by micro-CT and H&E staining. No significant difference in bone regeneration among the three groups in WT mice. Quantification of newly formed bone parameters from micro-CT and H&E staining showed that low-dose BMP2 treatment formed less new bone in ob/ob mice compared to WT. BMP2+low-dose leptin treatment substantially rescued the compromised bone regeneration in ob/ob mice up to the level in WT mice. However, the BMP2 and high dose of leptin failed to rescue the compromised bone regeneration in ob/ob mice. Our findings suggest that a combination of the low-dose BMP2 and leptin could be a strategy to promote osteogenesis in obese populations with leptin deficiency.

Leptin Modulates the mRNA Expression of Follicle Development Markers in Post-hatch Chicks in an Age-Dependent Manner.

Leptin is involved in regulating reproductive function in chickens, and the development of the leptin system is initiated during the early embryonic stage; however, whether leptin has a specific role in regulating the ovarian development in early post-hatch days is still not fully understood. This study investigated the expression of ovarian functional markers in growing juvenile chickens, along with the effects of leptin on gene expression in the hypothalamus–pituitary–gonadal (HPG) axis on specific ovarian-remodeling days. Leptin receptor (LEPR), follicle-stimulating hormone receptor (FSHR), and the mRNA expression of aromatase (CYP19A1) tended to increase with age in the ovaries of growing chicks. In the ovaries of 7-day-old chicks, intraperitoneally injected leptin significantly increased the mRNA expressions of LEPR, FSHR, and CYP19A1, and this resulted in the increased serum estradiol levels. However, leptin had no effect on hypothalamic LEPR, gonadotropin-releasing hormone 1 (GnRH1), or gonadotropin-inhibitory hormone (GnIH) mRNAs; however, in the pituitary gland, leptin significantly increased the mRNA expression of luteinizing hormone beta subunit (LHB) but had no effect on the mRNA expression of follicle-stimulating hormone beta subunit (FSHB). In 28-day-old chicks, hypothalamic and pituitary mRNAs were unaffected by leptin administration, except hypothalamic LEPR mRNA that was upregulated by a high dose of leptin. In the ovary, leptin dose-dependently decreased the mRNA expression of LEPR; low doses of leptin significantly increased the mRNA expression of FSHR, whereas high doses significantly decreased this expression; leptin did not affect the mRNA expression of CYP19A1; and high leptin doses significantly reduced the serum estradiol levels. Collectively, the results of this study show that leptin modulates ovarian development and folliculogenesis marker genes by primarily acting on ovaries on the specific ovarian-remodeling days in post-hatch chicks, which may alter folliculogenesis and ovarian development toward puberty in chicken.

The effect of intra-amygdalar leptin administration on anxiety, depression and learning behaviors in rats

Leptin is released by adipose tissue. Leptin can cross the blood–brain barrier and bind to receptors on neurons in brain areas to exert its biological function when released into circulation. This study aimed to determine the influences of intra-amygdalar administration of high and low doses of leptin on anxiety, depression, learning behaviors of rats. In the experimental protocol I, intra-amygdalar injection of high and low doses of leptin (0.1 and 1 μg/ kg) and saline were administered 30 min before the behavioral tests. Then, the animals were exposed to open field, elevated plus maze, Porsolt and Morris water maze tests for measuring of behaviors. In experimental protocol 2, the cerebrospinal fluids of all groups of experimental protocol 1 were collected by microdialysis method and then were analyzed by HPLC. The effect of the low dose of leptin was significant on the open field. The effect of the high and low dose of leptin was significant on the elevated plus maze test. The effect of the low dose of leptin was significant on mobility in the center of the Porsolt. A high dose of leptin group had spent less time around the platform than controls in the Morris water maze test. HPLC analysis showed that the amount of serotonin and glutamate in the amygdala region increased after low dose leptin administration. Intra-amygdalar injection of low doses of leptin may decrease anxiety and depression-like behavior in rats by increasing serotonin and glutamate levels in the amygdala.

Leptin Promotes Prostate Cancer Proliferation and Migration by Stimulating STAT3 Pathway

To better understand the link between obesity and prostate cancer (PC) aggressiveness, we investigate the role of leptin, an obesity associated adipokine, and its receptor (Ob-R) in PC cells migration. The migration assay (Wound-healing) was used to study the leptin impact on DU-145 and PC3 cells lines. STAT3 activation was performed by Western Blot. E-cadherin expression was studied using fluorescence microscopy and Ob-R expression in PC and benign prostatic Hyperplasia (BPH) biopsies was assessed by RT-PCR. In this study we demonstrate that high dose of leptin promotes PC cells migration and EMT transition via the stimulation of STAT3 pathway. In addition, we report that although Ob-R mRNA is expressed by ADK and BPH resections biopsies, significant higher levels were observed for ADK patients. Finally, we found a positive association between Ob-R mRNA expression and worse PC prognosis. A better understanding of the molecular processes of leptin signaling is crucial for identifying appropriate approaches for treatment of obesity-related PC patients.

Effects of leptin on lipid metabolism and inflammatory factors in diabetic rats

Objective: To investigate the effects of leptin on glucose metabolism and related inflammatory factors in diabetic rats. Methods: Ten healthy male Wistar rats were randomly selected as the control group. Fifty rats were fed with high sugar and high fat diet and injected with streptozotocin (STZ, 25 mg/kg) intraperitoneally. They were randomly divided into model group, leptin low, middle and high dose group. The rats in the low, middle and high dose group were fed with leptin at the doses of 20, 50 and 100 μg/kg for 5 d respectively. Blood glucose (FBG) was measured by GOD-PAP method, insulin content (INS) was tested by radioimmunoassay, the serum levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL-C) and high density lipoprotein (HDL-C) were determined by automatic biochemical analyzer, the contents of malondialdehyde (MDA), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the expression of leptin in adipose tissue of diabetic rats. Results: Compared with the control group, the blood glucose levels of other groups were increased significantly (P＜0.01). Compared with the model group, the blood glucose levels of middle and high dose leptin rats decreased significantly (P＜0.05, P＜0.01). The insulin level of high dose leptin group decreased significantly (P＜0.01). There was no significant difference in FBG and INS among the three groups (P＞0.05). Compared with the model group, TC levels of middle and high dose leptin group were decreased significantly (P＜0.05, P＜0.01). TG and LDL-C levels of high dose leptin group were decreased significantly (P＜0.05), HDL-C level of high dose group was increased significantly (P＜0.01). Compared with different dose groups, the high dose of leptin (100 μg/kg) could decrease the levels of TC, TG and LDL-C, and increase the level of HDL-C, which was better than those of the middle and low dose of leptin (P＜0.05) Compared with the model group (52.27±10.93), the levels of leptin in low, middle and high dose group were (47.35±12.09), (44.68±10.23) and (40.13±9.87) respectively, which could be decreased by leptin in a dose-dependent manner. Conclusion: The abnormal secretion of leptin is one of the factors inducing diabetes mellitus. Under the intervention of a certain concentration of exogenous leptin (100 μg/kg), it can significantly reduce the level of MDA, TNF-α, and improve the level of IL-6. The mechanism may be closely related to the reduction of inflammatory response, oxidative stress and correction of dyslipidemia. Leptin also reduces the risk of disease progression in diabetes treatment.

Jejunal Insulin Signalling Is Increased in Morbidly Obese Subjects with High Insulin Resistance and Is Regulated by Insulin and Leptin.

Little is known about the jejunal insulin signalling pathways in insulin resistance/diabetes states and their possible regulation by insulin/leptin. We study in jejunum the relation between insulin signalling and insulin resistance in morbidly obese subjects with low (MO-low-IR) or with high insulin resistance (MO-high-IR), and with type 2 diabetes treated with metformin (MO-metf-T2DM), and the effect of insulin/leptin on intestinal epithelial cells (IEC). Insulin receptor substrate-1 (IRS1) and the catalytic p110β subunit (p110β) of phosphatidylinositol 3-kinase (PI3K) were higher in MO-high-IR than in MO-low-IR. The regulatory p85α subunit of PI3K (p85α)/p110β ratio was lower in MO-high-IR and MO-metf-T2DM than in MO-low-IR. Akt-phosphorylation in Ser473 was reduced in MO-high-IR compared with MO-low-IR. IRS1 and p110-β were associated with insulin and leptin levels. The improvement of body mass index (BMI) and HOMA-IR (homeostasis model assessment of insulin resistance index) after bariatric surgery was associated with a higher IRS1 and a lower p85α/p110β ratio. IEC (intestinal epithelial cells) incubation with a high glucose + insulin dose produced an increase of p85α and p110β. High dose of leptin produced an increase of IRS1, p85α and p110β. In conclusion, despite the existence of insulin resistance, the jejunal expression of genes involved in insulin signalling was increased in MO-high-IR. Their expressions were regulated mainly by leptin. IRS1 and p85α/p110β ratio was associated with the evolution of insulin resistance after bariatric surgery.

Activation of the leptin pathway by high expression of the long form of the leptin receptor (Ob-Rb) accelerates chondrocyte senescence in osteoarthritis.

ObjectivesActivation of the leptin pathway is closely correlated with human knee cartilage degeneration. However, the role of the long form of the leptin receptor (Ob-Rb) in cartilage degeneration needs further study. The aim of this study was to determine the effect of increasing the expression of Ob-Rb on chondrocytes using a lentiviral vector containing Ob-Rb.MethodsThe medial and lateral cartilage samples of the tibial plateau from 12 osteoarthritis (OA) patients were collected. Ob-Rb messenger RNA (mRNA) was detected in these samples. The Ob-Rb-overexpressing chondrocytes and controls were treated with different doses of leptin for two days. The activation of the p53/p21 pathway and the number of senescence-associated β-galactosidase (SA-β-gal)-positive cells were evaluated. The mammalian target of rapamycin (mTOR) signalling pathway and autophagy were detected after the chondrocytes were treated with a high dose of leptin.ResultsIn total, 12 cases were found to have severe medial cartilage wear compared with the lateral cartilage. Immunofluorescence showed that the expression of Ob-Rb in the medial cartilage of the tibial plateau was high. High levels of leptin led to cell cycle arrest and inhibited autophagy. After overexpression of Ob-Rb, the physiological dose of leptin induced cell senescence in the chondrocytes. High doses of leptin inhibited autophagy by activating the mTOR signalling pathway. Blockade of the mTOR signalling pathway could restore autophagy and partially reverse senescence induced by leptin in chondrocytes.ConclusionIn summary, the present study demonstrated that high doses of leptin induce cell senescence by activating the mTOR pathway in chondrocytes from OA cartilage. Highly expressed Ob-Rb accelerates chondrocyte senescence by activating the leptin pathway in OA.Cite this article: X. Zhao, P. Huang, G. Li, L. Zhendong, G. Hu, Q. Xu. Activation of the leptin pathway by high expression of the long form of the leptin receptor (Ob-Rb) accelerates chondrocyte senescence in osteoarthritis. Bone Joint Res 2019;8:425–436. DOI: 10.1302/2046-3758.89.BJR-2018-0325.R2.

Sex- and bone-specific responses in bone structure to exogenous leptin and leptin receptor antagonism in the ovine fetus.

Widespread expression of leptin and its receptor in developing cartilage and bone suggests that leptin may regulate bone growth and development in the fetus. Using microcomputed tomography, this study investigated the effects of exogenous leptin and leptin receptor antagonism on aspects of bone structure in the sheep fetus during late gestation. From 125 to 130 days of gestation (term ~145 days), chronically catheterized singleton sheep fetuses were infused intravenously for 5 days with either saline (0.9% saline, n = 13), recombinant ovine leptin at two doses (0.6 mg·kg-1·day-1 LEP1, n = 10 or 1.4 mg·kg-1·day-1 LEP2, n = 7), or recombinant superactive ovine leptin receptor antagonist (4.6 mg·kg-1·day-1 SOLA, n = 6). No significant differences in plasma insulin-like growth factor-I, osteocalcin, calcium, inorganic phosphate, or alkaline phosphatase were observed between treatment groups. Total femur midshaft diameter and metatarsal lumen diameter were narrower in male fetuses treated with exogenous leptin. In a fixed length of femur midshaft, total and bone volumes were reduced by the higher dose of leptin; nonbone space volume was lower in both groups of leptin-treated fetuses. Leptin infusion caused increments in femur porosity and connectivity density, and vertebral trabecular thickness. Leptin receptor antagonism decreased trabecular spacing and increased trabecular number, degree of anisotrophy, and connectivity density in the lumbar vertebrae. The increase in vertebral porosity observed following leptin receptor antagonism was greater in the malecompared with female, fetuses. Therefore, leptin may have a role in the growth and development of the fetal skeleton, dependent on the concentration of leptin, sex of the fetus, and bone type examined.

Leptin and camel milk abate oxidative stress status, genotoxicity induced in valproic acid rat model of autism.

The aspect of treatment of autistic behaviour was investigated using valproic acid rat model of pregnant female rats. Two main groups (10 male rats/group) were treated for 6 days and then divided into six subgroups. The first group of normal rats was divided into three subgroups: (A) – control group, (B) – treated with camel milk (CAM; 2 mL/p.o) and (C) – treated with leptin (1000 µg/kg i.p) twice daily. The second group of autistic rats was randomly distributed into four subgroups as follows: (D) – positive control (autistics rats), (E) – treated with CAM, (F) – treated with a moderate dose of leptin and (G) – treated with a higher dose of leptin. Autistic behaviours of male offspring were checked by grooming and elevated pulz maze tests. Valproic acid (VPA)-induced autistic rats showed severe changes in oxidative stress markers, neurotransmitters and inflammatory cytokines, besides genotoxic manifestation of expression of tumour necrosis factor (TNF)-α, Bax and caspase-3. Leptin or CAM alone showed no signs of toxicity. CAM showed pronounced improvement in control rats than control itself. Leptin or CAM treatment of autistic animals showed a significant improvement of all measured parameters and genetic expression values. The improvement was pronounced in animals treated with CAM. These results suggest that CAM is a potential therapeutic candidate for autism via regulation of inflammatory and apoptotic pathways. Leptin plays an essential role in alleviation of autistic behaviour through antioxidant effects.

In ovo leptin administration modulates glucocorticoid receptor mRNA expression specifically in the hypothalamus of broiler chickens

The glucocorticoid receptor (GR) is well documented to play a crucial role in the central control of energy homeostasis in mammals. However, the distribution and function of the GR in the chicken brain are less clear. Leptin is a key hormone regulating energy homeostasis in mammals, yet its action in the chicken is still under debate. In this study, the distribution of GR mRNA in the chicken brain and the effects of in ovo administration of leptin and its antagonist on early post-hatch growth and GR mRNA expression in different hypothalamic nuclei were investigated via in situ hybridization (ISH) and quantitative PCR. GR mRNA was widely expressed in the chicken brain, mainly in the corpus striatum, nucleus rotundus, dorsolateral nucleus, nucleus ovoidalis, nucleus reticularis superior and the hippocampus (Hp) and in the preoptic area of the hypothalamus. High doses of leptin (5.0μg) significantly promoted post-hatch growth, resulting in a significant high body weight increased by 24.64% at day (D) 21 of life. Meanwhile, hypothalamic expression of GR mRNA in the LL and HL groups was down-regulated significantly by 7.02% and 13.65% respectively (P<0.05). ISH revealed region-specific changes: GR mRNA was found to be significantly decreased (P<0.05) in the paraventricular nucleus, periventricular nucleus and ventromedial nucleus but not in the Hp, infundibular nucleus or lateral hypothalamus of D21 broiler chickens. The leptin antagonist was able to reverse the effect of leptin on the growth rate and hypothalamic GR mRNA expression. These results provide evidence that in ovo administration of leptin influences early post-hatch growth and the hypothalamic expression of GR mRNA in broiler chickens.

Jejunal gluconeogenesis associated with insulin resistance level and its evolution after Roux-en-Y gastric bypass

BackgroundIntestinal gluconeogenesis (GNG) may play an important role in glucose homeostasis, but there is little information about the condition in humans. ObjectivesTo study the relationship between intestinal GNG and insulin resistance, its association with the evolution of morbidly obese patients after bariatric surgery, and the effect of insulin and or leptin. SettingRegional university hospital, Malaga (Spain). MethodsJejunal mRNA expression of genes involved in GNG was analyzed in 3 groups of morbidly obese patients who underwent Roux-en-Y gastric bypass: with low insulin resistance (MO-low-IR), with high insulin resistance (MO-high-IR), and with type 2 diabetes treated with metformin (MO-metf-T2D). Also, intestinal epithelial cells (IEC) from MO-low-IR were incubated with different doses of insulin and or leptin. ResultsIn MO-high-IR, glutaminase, phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6 Pase), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 α), and sterol regulatory element-binding proteins 1 c (SREBP-1 c) expressions were significantly higher than in MO-low-IR. In MO-metf-T2 D, only PEPCK was significantly lower than in MO-high-IR. In IEC, an incubation with a high glucose and insulin dose produced an increase of PEPCK and SREBP-1 c, and a decrease of glutaminase, fructose 1,6-bisphosphatase (FBPase), and PGC-1 α expression. At high doses of leptin, G6 Pase and FBPase were significantly increased. The improvement of insulin resistance 3 months after bariatric surgery was positively associated with high G6 Pase and FBPase expression. ConclusionmRNA expression of genes involved in GNG is increased in the jejunum of MO-high-IR, and regulated by insulin and or leptin. High mRNA expression of genes involved in GNG is associated with a better evolution of insulin resistance after bariatric surgery.

Fourth-ventricle leptin infusions dose-dependently activate hypothalamic signal transducer and activator of transcription 3.

Previous studies have shown that very low-dose infusions of leptin into the third or the fourth ventricle alone have little effect on energy balance, but simultaneous low-dose infusions cause rapid weight loss and increased phosphorylation of STAT3 (p-STAT3) in hypothalamic sites that express leptin receptors. Other studies show that injecting high doses of leptin into the fourth ventricle inhibits food intake and weight gain. Therefore, we tested whether fourth-ventricle leptin infusions that cause weight loss are associated with increased leptin signaling in the hypothalamus. In a dose response study 14-day infusions of increasing doses of leptin showed significant hypophagia, weight loss, and increased hypothalamic p-STAT3 in rats receiving at least 0.9 μg leptin/day. In a second study 0.6 μg leptin/day transiently inhibited food intake and reduced carcass fat, but had no significant effect on energy expenditure. In a final study, we identified the localization of STAT3 activation in the hypothalamus of rats receiving 0, 0.3, or 1.2 μg leptin/day. The high dose of leptin, which caused weight loss in the first experiment, increased p-STAT3 in the ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus. The low dose that increased brown fat UCP1 but did not affect body composition in the first experiment had little effect on hypothalamic p-STAT3. We propose that hindbrain leptin increases the precision of control of energy balance by lowering the threshold for leptin signaling in the forebrain. Further studies are needed to directly test this hypothesis.

Experimental study on precaution osteoporosis by the increase of cerebrospinal fluid leptin level on rabbit model

Objective To explore effect of increasing cerebrospinal fluid (CSF) leptin level by administration from the foramen magnum to the cerebellum medulla oblongata pool in osteoporosis in rabbit model established by ovariectomy (OVX) and methylprednisolones. Methods According to certain inclusion and exclusion criteria, 30 adult rabbits which were healthy 5-month-old female New Zealand white rabbits with the same batch and weight were divided into 5 groups (n=6) with random number table, including control group, normal saline group, Low dose group, middle dose group and high dose group. The animal model of osteoporosis (OP) was established 8 weeks after ovariectomy (bilateral ovarian resection) and intramuscular injection of methylprednisolone daily for 8 consecutive weeks in all groups. The control group were only simulated puncture and didn’t inject any drugs or normal saline, the normal saline group were intracerebroventricular (ICV) injection with NS and Low, Middle and High dose group injected with low, middle and high dose of leptin respectively at 0,3,7 days after OVX. Before (0 week) and 4,8 weeks after the operation, the measure of dual energy X-ray absorptiometry was done on all the rabbits femur and bone mineral density (BMD) was delivered. Before (0 week) and 4, 8 weeks after the first administration, leptin in cerebrospinal fluid (CSF) and serum growth hormone (GH), insulin growth factor 1 (IGF-1) and osteocalcin (OT/BGP) were tested with Enzyme linked immunosorbent assay and serum calcium, phosphorus and alkaline phosphatase (ALP) with automatic biochemical analyzer (ACA). The rabbits were sacrificed and the thalamus and femoral neck were harvested to test the expression of leptin receptor (LEPR) in thalamus and bone morphogenetic protein-2 (BMP-2) and typeⅠcollagen (ColⅠ) at 8 weeks after the first administration. Results Compared with the control group and NS group, the cerebrospinal fluid (CSF) leptin concentration of leptin administration groups kept in a higher level significantly at 4 and 8 weeks after leptin administration, it was positively correlated with the concentration of administration. After administration 4 and 8 weeks, the bone mineral density (BMD) of leptin administration groups were not significantly lower than that of non-administration groups, and the high dose group was unchanged. The serum osteocalcin/bone glad protein (OT/BGP) concentration of leptin administration groups decreased more significantly compared with the non-administration groups at 4 weeks after leptin administration; and it rebounded more significantly than the non-administration groups and low dose group rebounded more significantly than other non-administration groups at 8 weeks. The concentration of serum growth hormone (GH) of 5 groups showed a downward trend at 4 and 8 weeks, the serum GH concentration of leptin administration groups decreased more significantly than the non-administration groups. The concentration of serum insulin-like growth IGF-1 of 5 groups were decreased significantly at 4 weeks, and the serum IGF-1 concentration of control and low dose groups were significantly higher than other 3 groups at 8weeks. Compared with the non-administration groups, the concentration of serum Calcium of leptin administration groups were increased more obvious, and it was positively correlated with the concentration of administration. Serum phosphorus of 5 groups were decreased at 4 weeks and elevated at 8 weeks; there was no difference among the groups. Serum ALP showed a continuous downward trend, and the middle dose group decreased significantly compared with other groups at 4 and 8 weeks. RT-PCR results showed that the BMP-2 and Col ⅠmRNA expression increased and positively correlated with leptin administration concentration at 8weeks. Conclusion The increase of cerebrospinal fluid leptin level can promote the bone metabolism, reduce bone loss and precaution the occurrence of osteoporosis in rabbit model caused by OVX and methylprednisolone, and it was positively correlated with the concentration of administration. That provides a new method for the prevention and treatment of osteoporosis in rabbit model. Key words: Leptin; Osteoporosis; Bone density

The effect of leptin treatment on in vivo cardiac parameters of streptozotocin induced diabetic rats

Aims Diabetes mellitus is a chronic metabolic disease, which results in serious cardiac complications. It has been shown that hemodynamic parameters are impaired in diabetic rat heart. Leptin is a hormon secreted from adipose tissue and have important effects on glucose metabolism. The effect of leptin treatment on cardiac hemodynamic parameters in streptozotocine (STZ) induced diabetic rat heart has not been studied yet. Thus, we aimed to investigate whether leptin treatment could ameliorate impaired basal cardiac hemodynamic parameters in STZ diabetic rat. Methods Male Sprague-Dawley rats were used for this study. The rats were divided into 3 groups; control (C), diabetic (D), leptin treated diabetic group (Lep). Diabetes was induced by single dose streptozotocin injection (40 mg/kg, i.v.). At the end of 10-week-diabetes period some of the diabetic rats were treated with leptin (150 μg/kg/day, via osmotic minipump) for 14 days. In vivo cardiac hemodynamic parameters were determined through pressure-volume (PV loop) relations. First pressure in carotid artery [systolic arterial pressure (SP), diastolic arterial pressure (DP), mean arterial pressure (MAP)] were measured, then PV loops were recorded under isofluoran anesthesia (%2). Cardiac hemodynamic parameters include heart rate (HR), end systolic pressure (ESP), end diastolic pressure (EDP), the rate of contraction and relaxation (dp/dtmax and dp/dtmin), end systolic volume (ESV), end diastolic volume (EDV), stroke volume (SV), cardiac output (CO), ejection fraction (EF), isovolumic relaxation constant (Tau, Weiss and glantz method), end systolic elastance (Ees), cardiac stiffness, preload recruitable stroke work (PRSW) and dp/dt-EDV relation. To assess preload recruitable cardiac parameters inferior vena cava occlusion was made. Results PV loop analysis showed that almost all cardiac hemodynamic parameters were altered in diabetic state as expected. Heart rate of rats were decreased in D group, leptin treatment did not improve it significantly. ESP values were decreased in D group. Leptin treatment increased it markedly, however it did not reach control levels. EDP values did not differ between the groups. Both dp/dtmax and dp/dtmin values were significantly attenuated in D group, leptin treatment ameliorated it markedly. EDV values were enhanced in D group, and decreased after leptin treatment. There were no significant changes in SV between the groups. CO was found to be attenuated in D group. EF values were not changed. Tau values were increased markedly in D group, and improved with leptin treatment, however it was still high compared to C group. Ees was decreased in D group. No significant change was seen after leptin treatment. The stiffness of the left ventricle was increased in D group and decresed after leptin treatment. PRSW was attenuated in D group and leptin treatment improved it markedly. dp/dt-EDV relation was also decreased in D group and ameliorated partly in Lep group. SP, DP and MAP were decreased in D group and ameliorated in Lep group. Conclusion In vivo cardiac experiments showed that heart function was impaired in diabetic rats. Leptin exerted beneficial effects on these parameters, however the values did not reach control levels. This may be related to chosen dose of leptin, which has been demonstrated effective in a previous study. To confirm the dose dependency of the effect, cardiac parameters should be evaluated in rats treated with a higher dose of leptin.

Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation.

Leptin is a hormone produced by adipose tissue that regulates various physiological processes. Recent studies have shown that the level of circulating leptin is elevated in obese patients and have suggested a relationship between obesity and postoperative lymphedema. However, the mechanisms by which postoperative lymphedema develops in obese patients and the mechanisms by which leptin regulates lymphatic endothelial cell homeostasis such as tube formation and cell proliferation remain unknown. Here we report that leptin regulates tube formation and cell proliferation in human dermal lymphatic endothelial cells (HDLECs) by activation of the signal transducer and activator of transcription 3 pathway, which is downstream signaling of the leptin receptor. Additionally, we found that upregulation of suppressor of cytokine signaling 3 underlies the mechanisms by which a high dose of leptin inhibits cell proliferation and tube formation. Leptin also enhanced expression of the proinflammatory cytokine IL-6 in HDLECs. Interestingly, IL-6 rescues the compromised cell proliferation and tube formation caused by treatment with a high dose of leptin in an autocrine or paracrine manner. Taken together, our findings reveal a novel mechanism by which compromised HDLECs maintain their homeostasis during inflammation mediated by leptin and IL-6. Thus, regulating the level of leptin or IL-6 may be a viable strategy to reduce the incidence of postoperative lymphedema.

Leptin changes differentiation fate and induces senescence in chondrogenic progenitor cells.

Body weight is a component of the mechanical theory of OA (osteoarthritis) pathogenesis. Obesity was also found to be a risk factor for digital OA involving non-weight-bearing joints, which suggested that metabolism influences the occurrence and progression of OA. The metabolic origin of OA has been partially attributed to the involvement of adipokines, such as leptin, the levels of which are significantly and positively correlated with cartilage degeneration in OA patients. However, the mechanisms by which leptin-induced cartilage degeneration occurs are poorly understood. The discovery of chondrogenic progenitor cells (CPCs) opened up new opportunities for investigation. Investigating the effects of leptin on differentiation and proliferation in CPCs would increase our understanding of the roles played by leptin in the aetiology and development of OA. Here, CPCs were harvested using single-cell sorting from rat cartilage tissues to obtain mesenchymal stem-like cells, which possess clonogenicity, proliferation and stemness. High doses of leptin decreased the ability of the CPCs to migrate, inhibited their chondrogenic potential and increased their osteogenic potential, suggesting that leptin changes differentiation fates in CPCs. High doses of leptin induced cell cycle arrest and senescence in CPCs by activating the p53/p21 pathway and inhibiting the Sirt1 pathway. Inhibiting the Sirt1 pathway accelerated cartilage senescence in knockout (KO) mice. Activating the leptin pathway induced higher Ob-Rb expression and was significantly correlated with cartilage degeneration (lower levels of Coll-2) and tissue senescence (higher levels of p53/p21 and lower levels of Sirt1) in OA patients, suggesting that leptin-induced CPCs senescence contributes to the development of OA. Taken together, our results reveal new links between obesity and cartilage damage that are induced by leptin-mediated effects on cell behaviour and senescence.

Behavioral, hormonal and central serotonin modulating effects of injected leptin

Leptin is viewed as an important target for developing novel therapeutics for obesity, depression/anxiety and cognitive dysfunctions. The present study therefore concerns behavioral, hormonal and central serotonin modulating effects of systemically injected leptin. Pharmacological doses (100 and 500μg/kg) of leptin injected systemically decreased 24h cumulative food intake and body weight in freely feeding rats and improved acquisition and retention of memory in Morris water maze test. Potential anxiety reducing, hormonal and serotonin modulating effects of the peptide hormone were determined in a separate experiment. Animals injected with 100 or 500μg/kg leptin were tested for anxiety in an elevated plus maze test 1h later. A significant increase in the number of entries and time passed in open arm of the elevated plus maze in leptin injected animals suggested pronounced anxiety reducing effect. Moreover, circulating levels of leptin correlated significantly with anxiety reducing effects of the peptide hormone. Serum serotonin increased and ghrelin decreased in leptin injected animals and correlated, positively and negatively respectively, with circulating leptin. Corticosterone increased at low dose and levels were normal at higher dose. Serotonin metabolism in the hypothalamus and hippocampus decreased only at higher dose of leptin. The results support a role of leptin in the treatment of obesity, anxiety and cognitive dysfunctions. It is suggested that hormonal and serotonin modulating effects of leptin can alter treatment efficacy in particularly comorbid conditions.

Upregulated Leptin in Periodontitis Promotes Inflammatory Cytokine Expression in Periodontal Ligament Cells.

Imbalance or disruption in the expression of inflammatory mediators contributes greatly to the breakdown of the periodontal supporting tissues. Leptin, through binding to its receptor (obesity-related leptin and leptin receptor [OBR]), has potent effects on immunity and inflammation. However, to date, researchers only indicated a role of leptin in periodontitis. No direct or valid evidence exists about how leptin and its receptor are regulated by local inflammation, what effects they have, and the underlying mechanisms. Experimental periodontitis was induced by ligation of mandibular second molars in beagle dogs. The expression of leptin, OBR, and interleukin (IL)-1β was examined by immunohistochemistry. Meanwhile, recombinant human IL-1β was used to stimulate human periodontal ligament cells (hPDLCs) in vitro, and mRNA and protein levels of leptin were measured using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, mRNA and protein levels of IL-6 and IL-8 were measured using real-time PCR and ELISA, after stimulation with various concentrations of leptin, knocking down all or only the long form of OBR (OBRb) by small interfering RNA and incubation with multiple intracellular signaling pathway inhibitors, respectively. Leptin and OBR increased substantially in inflammatory periodontal tissues, which correlated well with the extent of inflammatory infiltration, and was a result of the upregulation in resident cells themselves. A high dose of leptin could induce the expression of mRNA and protein of IL-6 and IL-8 in hPDLCs through binding with OBRb and activating different intracellular signaling pathways. Upregulated leptin and OBR in periodontitis stimulated proinflammatory cytokine expression in PDL cells to additionally promote local inflammation.