Low Dose Of Leptin Research Articles

(063) The Role of Leptin as a Testicular Immune Microenvironment Modulator and Leydig Stem Cell Function

Abstract Introduction The testicular microenvironment, consisting of cells including Sertoli and peritubular cells, releases paracrine factors that may regulate testicular function. In previous research, Leptin was identified as a critical paracrine factor in the testicular microenvironment. Leptin is secreted by Sertoli and peritubular cells and binds to its receptor in Leydig cells. This interaction triggers the expression of the desert hedgehog signaling pathway, which is essential for Leydig stem cell differentiation into adult Leydig cells, which are capable of producing testosterone. However, our earlier study had a limitation: the human cell model used was derived from testes biopsies, lacking an intact immune microenvironment. This study investigates the effect of Leptin on a complete testicular immune microenvironment in a murine model. Objective To investigate the impact of Leptin on a complete testicular immune microenvironment. Methods We used 6-week-old C57BL6 mice, exposed to two different concentrations of Leptin, 10 and 100 mg/ml, for seven days. After the treatment period, the animals were humanely euthanized, and their blood and testes were collected. Blood samples underwent a complete blood count differential profile analysis using a Hemavet Mascot Multispecies Hematology System Counter 1500R. Testes samples were subjected to comprehensive immunophenotyping, allowing us to assess the impact of Leptin treatment on various immune cell types within the testes Results Leptin at a low dose (10 mg/ml) showed increased, but not significant, levels of serum testosterone (13.20 + 0.37 ng/dl), while LH and FSH levels significantly (p<0.05) increased. In contrast, animals receiving 100 mg/ml Leptin showed no significant changes in hormone levels. CBC profiling indicated that lymphocytes and platelets were significantly influenced, specifically with low doses of Leptin. Comprehensive immunophenotyping revealed a significant increase in CD4+ (helper T cells) and CD8+ (cytotoxic T lymphocytes) cell numbers in animals treated with low doses of Leptin, a pattern not observed with high doses. Conclusions This study uncovers previously unexplored aspects of how paracrine factors, released by Sertoli and peritubular myoid cells, can modulate the immune microenvironment within the testis, potentially impacting testosterone production. Future research will investigate the relationship between changes in immune cell types, increased T cell numbers, and their influence on Leydig stem cell differentiation into adult Leydig cells Disclosure No.

(251) Exogenous Leptin Treatment Augments Testicular T-cell Secretion and Function Differentially in Context to BMI in Murine Models

Abstract Introduction In previous research, we explored the effects of exogenous Leptin treatment on modulating the immune landscape within the testicular microenvironment (TME). We found that within the TME, it is the CD8+ T cells which were enriched upon Leptin treatment. However, our earlier study did not took into account the impact of variations in BMI which could potentially confound the effects of Leptin on T cells and on TME as a whole. Objective To investigate the impact of Leptin on a complete testicular immune microenvironment. Methods We used 6-week-old C57BL6 mice (n=30), grouped into three, with first group subjected to regular diet, second to Lean fat and third to high fat inducing diet. Once the mice reached the desired weights, they were exposed to two different concentrations of Leptin, 10 and 100 mg/ml, for seven days. After the treatment period, the animals were humanely euthanized, and their blood and testes were collected. Blood samples underwent a CBC differential profile analysis using a Hemavet Mascot Multispecies Hematology System Counter 1500R. Testes samples were subjected to comprehensive immunophenotyping, allowing us to assess the impact of Leptin treatment on various immune cell types within the testes. Results In mice with regular BMI, Leptin at a low dose (10 mg/ml) showed increased, but not significant, levels of serum testosterone (13.20 + 0.37 ng/dl), while LH and FSH levels significantly (p<0.05) increased. In contrast, animals receiving 100 mg/ml Leptin showed no significant changes in hormone levels. CBC profiling indicated that lymphocytes and platelets were significantly influenced, specifically upon low doses of Leptin. Comprehensive immunophenotyping revealed a significant increase in CD4+ (helper T cells) and CD8+ (cytotoxic T lymphocytes) cell numbers in animals treated with low doses of Leptin, a pattern not observed with high doses. These results were in contrast to the mice which represented lean and obese conditions. Conclusions This study uncovers previously unexplored aspects of how exogenous Leptin treatments can modulate the immune microenvironment within the testis, potentially impacting testosterone production. Future research will investigate the relationship between changes in immune cell types, increased T cell numbers, and their influence on Leydig stem cell differentiation into adult Leydig cells. Disclosure No.

(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

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.

Abstract 436: Administering Leptin into the Rostral Ventral Lateral Medulla (RVLM) Augments Blood Pressure and Renal Sympathetic Nerve Activity

Initially described as a hormone that regulates energy homeostasis, we now know that leptin is an integrative hormone that responds to and regulates various systems, in particular, the renal sympathetic nervous system. The rostral ventral lateral medulla (RVLM) contains neurons which evoke changes of arterial blood pressure by controlling the tonic and phasic activation of sympathetic activity. Thus, we sought to determine if leptin regulates blood pressure and renal sympathetic activity within the RVLM. Utilizing immunohistochemistry, we observed that neurons within the RVLM that receive sympathetic projections from the kidney express leptin receptors on them. We have also demonstrated that these neurons are responsive to low doses of leptin and evoke significant increases in blood pressure, renal sympathetic nerve activity and heart rate. Taken together, these data demonstrate that leptin can regulate blood pressure and renal sympathetic nerve activity within the RVLM.

In ovoleptin administration accelerates post‐hatch muscle growth and changes myofibre characteristics, gene expression and enzymes activity in broiler chickens

To evaluate the effect of maternal leptin on muscle growth, we injected 0 μg (control, CON), 0.5 μg (low leptin dose, LL) or 5.0 μg (high leptin dose, HL) of recombinant murine leptin dissolved in 100 μl of PBS into the albumen of broiler eggs prior to incubation. The newly hatched chicks were all raised under the same conditions until 21 days of age (D21), when body weight was measured and samples of gastrocnemius muscle were collected and weighed. Myosin ATPase staining was applied to identify myofibre types and measure the cross-sectional area (CSA) of myofibres. Real-time PCR was performed to quantify leptin receptor (LEPR), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor (GHR) and myostatin (MSTN) mRNA expression in the gastrocnemius muscle. The activity of calpains (CAPNs) in the gastrocnemius muscle was measured using a quantitative fluorescence detection kit. Male chickens treated with both high and low doses of leptin had significantly higher (p < 0.05) body weight on D21. The high leptin significantly increased the CSA (p < 0.05) of gastrocnemius muscle in male chickens, which coincided with a 93% increase (p < 0.05) in IGF-1 mRNA expression. Likewise, the LL dose increased the weight of gastrocnemius muscle in male chickens (p < 0.05), which was accompanied by a 41% down-regulation (p < 0.05) of MSTN mRNA expression and a decreased activity of CAPNs. However, all these changes were not observed in female chickens. The proportion of myofibre types did not altered. No significant change was detected for LEPR and GHR mRNA expression. These results indicate that in ovo leptin treatment affects skeletal muscle growth in chickens in a dose-dependent and sex-specific manner. The altered expression of IGF-1, MSTN mRNA and activity of CAPNs in skeletal muscle may be responsible for such effects.

Physiological Low Doses of Leptin and Cholecystokinin Induces Body Weight-Loss in Juvenile and Lean, but not in Adult-Obese Rats

Leptin and cholecystokinin (CCK) are two hormones involved in body weight regulation and feeding behavior. Leptin, an adiposity-derived signal, and CCK, a gut-satiety signal, interact in a synergistic manner and their concomitant treatment causes more weight loss than any hormone administered alone. The synergistic interaction between CCK and leptin has been widely characterized in lean rats, but there are a few studies performed in obese rats. Herein, in the same individual, we compared the sensitivity to CCK, leptin and the combined treatment, leptin + CCK, on body weight and food intake in two experiments one performed in juvenile-lean rats, and in a second experiment comparing adult Chow-fed versus adult-high fat diet (HFD) rats. We found that only the combined treatment had a long-term effect on food intake and caused a substantial and synergistic body weight loss, in juvenile rats. However, the same combined treatment failed to induce weight loss in both Chow-fed and HFD rats. We thus conclude that the sensitivity to leptin + CCK was age dependent, and at the doses used ineffective in adult and obese rats.

Body Fat as a Regulator of Bone Mass: Experimental Evidence from Animal Models

Animal models have proven valuable for elucidating the effects of body fat on bone metabolism, and this review highlights recent findings relevant for better understanding the relationship between adiposity and bone mass. Genetically obese mice and rats with altered leptin signaling consistently show reduced bone mass, demonstrating that leptin deficiency can contribute directly to osteopenia; however, the effect of leptin on bone is concentration dependent, such that relatively low doses of leptin stimulate bone formation, whereas high concentrations of leptin suppress bone formation and increase bone resorption. The hyperleptinemia associated with obesity and leptin receptor downregulation is therefore likely to have a negative impact on bone modeling and adaptation. Likewise, diets high in fat and sugar have been observed to increase bone fragility and decrease the accumulation of bone mineral in growing rodents. In vitro studies reveal that increasing concentrations of glucose and lipid can inhibit osteogenic differentiation of mesenchymal stem cells (MSCs), and instead direct these precursors toward the adipocyte lineage. Taken together, the evidence from animal studies indicates that excess body fat and obesogenic diets may attenuate the accretion of skeletal mass in growing animals, suggesting that low bone mass (relative to body weight) may be a potential side-effect of pediatric obesity.

Exogenous leptin promotes the recovery of regressed ovary in fasted ducks

The present study was undertaken to examine the effect of administered recombinant mouse leptin on the recovery of regressed ovary in fasted ducks. Twenty-eight ducks were divided into five groups: fed ad libitum (control; n = 5), fasted control (FC; n = 5), fasted + low dose of leptin (F + L; n = 5), fasted + medium dose of leptin (F + M; n = 5) and fasted + high dose of leptin (F + H; n = 3). All four fasted groups were fasted for 2 days and then ad libitum and the ducks were treated with leptin at doses of 0 (control and FC), 50 (F + L), 250 (F + M) and 1000 (F + H) μg/kg body weight/day on day 3–5. Results showed that a moderate dose of leptin (250 μg/kg body weight/day) injected during the re-feeding period: (i) promoted the recovery of the regressed ovary as evidenced by an increase in ovary weight and recovery of yellow hierarchical follicles; (ii) elevated the plasma 17β-estradiol (E 2) level; (iii) increased the mRNA levels of ovary follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and estrogen receptor-β (ER-β). Furthermore, the results also showed that a high dose of leptin (1000 μg/kg body weight/day) may have a negative effect on the recovery of the regressed ovary. In conclusion, this study indicates that, in ducks, leptin may be involved in the recovery of the regressed ovary caused by 2 days of fasting. This effect may be related to increased plasma E 2 levels and stimulation of the mRNA levels of ovarian FSHR, LHR and especially ER-β.

Hindbrain leptin injections induce forebrain as well as hindbrain signaling.

Leptin injections into the lateral, third or fourth ventricles decrease 24 h food intake and body weight gain. When we injected leptin into either the fourth or lateral ventricles on three consecutive days, 24 h food intake and body weight were markedly reduced by comparable amounts in association with both routes of administration. As expected, reductions of food intake by lateral ventricle leptin were associated with decreased meal size. However, fourth ventricle leptin unexpectedly caused a reduction in meal number, but not meal size. We hypothesized that the two routes of administration might influence distinct but interconnected ingestive control circuits. In order to evaluate this hypothesis, we analyzed STAT3 phosphorylation in the dorsal hindbrain and in the hypothalamus after leptin injection into either fourth or lateral cerebral ventricle. Leptin increased phospho-STAT3 in hindbrain and hypothalamus, regardless of its site of injection. Because our ventricular injection volumes were relatively large (3 μl), we also injected low doses of leptin (200 ng) in relatively small volumes (200 nl) directly into the nucleus of the solitary tract (NTS). NTS leptin induced increased phosphorylated STAT3 in the hypothalamus as well as the hindbrain. These results suggest that local leptin administration evokes STAT3 phosphorylation both near the site of injection and at remote sites, such as the hypothalamus. The mechanisms by which NTS leptin evokes hypothalamic STAT3 phosphorylation are under investigation. Supported by NIH Grant NS20561 and Netherlands Organisation of Scientific Research (NWO Rubicon).

Biphasic effects of leptin in porcine granulosa cells.

The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

Effects of prolonged leptin infusion on rat pituitary-adrenocortical function

Compelling evidence suggests that a regulatory loop between leptin and the hypothalamo-pituitary-adrenal (HPA) axis is operative, where ACTH inhibits leptin secretion by adipose tissue and in turn leptin increases expression and secretion of ACTH. However, conflicting findings have been obtained in vivo on the acute and chronic effects of leptin on the HPA axis. Adult female Wistar rats, kept in metabolic cages, were intraperitoneally infused for 2, 4, 8 or 16 days with leptin (10 nmol/kg. 24 h); control animals were infused with the vehicle only. The rate of body-weight gain was similar in control and leptin-infused rats. At day 16 of treatment relative pituitary weight was higher and relative adrenal weight smaller in leptin-infused than control rats. Pituitary ACTH concentration gradually decreased with the duration of treatment, and the drop was significantly higher in leptin-infused than control rats. During the entire experimental period the blood level of aldosterone was similar in both groups of rats. Conversely, at days 2 and 4 of treatment the blood concentration of corticosterone was lower in leptin-infused than in control rats, which at these times displayed elevated levels of circulating corticosterone. Taken together, these findings allow us to conclude that in the rat the prolonged infusion of low doses of leptin i) primarily depresses pituitary ACTH production, the effect being probably mediated by the hypothalamus; and ii) inhibits corticosterone response to the stress evoked by placing of animals in the metabolic cages.

Insulin and leptin acutely regulate cholesterol ester metabolism in macrophages by novel signaling pathways.

Leptin is produced in adipose tissue and acts in the hypothalamus to regulate food intake. However, recent evidence also indicates a potential for direct roles for leptin in peripheral tissues, including those of the immune system. In this study, we provide direct evidence that macrophages are a target tissue for leptin. We found that J774.2 macrophages express the functional long form of the leptin receptor (ObRb) and that this becomes tyrosine-phosphorylated after stimulation with low doses of leptin. Leptin also stimulates both phosphoinositide 3-kinase (PI 3-kinase) activity and tyrosine phosphorylation of JAK2 and STAT3 in these cells. We investigated the effects of leptin on hormone-sensitive lipase (HSL), which acts as a neutral cholesterol esterase in macrophages and is a rate-limiting step in cholesterol ester breakdown. Leptin significantly increased HSL activity in J774.2 macrophages, and these effects were additive with the effects of cAMP and were blocked by PI 3-kinase inhibitors. Conversely, insulin inhibited HSL in macrophages, but unlike adipocytes, this effect did not require PI 3-kinase. These results indicate that leptin and insulin regulate cholesterol-ester homeostasis in macrophages and, therefore, defects in this process caused by leptin and/or insulin resistance could contribute to the increased incidence of atherosclerosis found associated with obesity and type 2 diabetes.

Inhibition of NO synthesis enhances chronic cardiovascular and renal actions of leptin.

Acute studies suggest that leptin has pressor and depressor actions, including stimulation of sympathetic activity as well as increased release of NO from the vascular endothelium. The goal of this study was to examine the role of NO in modulating the chronic blood pressure, heart rate, and renal responses to hyperleptinemia, comparable to that found in obesity-induced hypertension. Male Sprague-Dawley rats were implanted with arterial and venous catheters, and mean arterial pressure and heart rate were monitored continuously 24 h/d. After a 4-day control period, the rats were infused with isotonic saline vehicle (n=6) or N(G)-nitro-L-arginine methyl ester (L-NAME, 10 microgram/kg per minute; n=9) to inhibit NO synthesis for 7 days. After 7 days of vehicle or L-NAME administration, leptin was infused intravenously for 7 days at a rate of 0.5 microgram/kg per minute, followed by a leptin infusion at 1.0 microgram/kg per minute for 7 days, along with vehicle or L-NAME. A 21-day infusion of L-NAME alone (n=6) served as a control for the L-NAME+leptin rats. Although the low dose of leptin alone did not significantly elevate arterial pressure, it raised the heart rate by 18+/-3 bpm. The higher leptin infusion rate raised arterial pressure from 96+/-3 to 104+/-3 mm Hg but did not increase the heart rate further. L-NAME+leptin increased arterial pressure by 40+/-6 mm Hg and heart rate by 79+/-19 bpm compared with pretreatment levels. In control L-NAME rats, mean arterial pressure increased by 31+/-4 mm Hg, whereas the heart rate was not altered significantly compared with pretreatment levels. Neither chronic leptin infusion alone nor L-NAME alone altered the glomerular filtration rate or renal plasma flow significantly, but L-NAME+leptin reduced glomerular filtration rate by 27+/-11% and renal plasma flow by 47+/-9%. These results indicate that impaired NO synthesis mildly enhances the chronic renal hemodynamic and hypertensive effects of leptin but markedly amplifies the tachycardia caused by hyperleptinemia.

Role of Nitric Oxide in Modulating the Chronic Cardiovascular and Renal Actions of Leptin

64 Acute studies suggest that leptin has pressor and depressor actions, including stimulation of sympathetic activity as well as increased release of nitric oxide (NO)from the vascular endothelium. The goal of this study was to examine the role of NO in modulating the chronic blood pressure, heart rate, and renal responses to hyperleptinemia, comparable to that found in obesity hypertension. Male Sprague-Dawley rats were implanted with arterial and venous catheters and mean arterial pressure (MAP) and heart rate (HR) were monitored continuously 24h/d. After a 4 day control period, rats were treated with vehicle (n=6) or L-NAME (n=7) for 7 days. After 7 days of vehicle or L-NAME (10 μg/kg/min) treatment to inhibit NO synthesis, rats received a low rate (0.5 μg/kg/min) leptin infusion for 7 days followed by a moderate rate (1.0 μg/kg/min) leptin infusion for 7 days, along with vehicle or L-NAME. A 21 day infusion of L-NAME alone (n=6) served as a control for the leptin+L-NAME rats. The low and moderate leptin infusions reduced food intake by 53±2%and 51±7%, respectively, but L-NAME did not alter the anorectic effect of leptin. Although the low dose of leptin alone did not significantly elevate MAP, it raised HR by 18±3 bpm. The moderate dose of leptin raised MAP from 96±3 to 104±3, but did not increase HR further. In rats pre-treated with L-NAME, the moderate dose of leptin raised MAP by 40±5 mmHg and HR by 73±17 bpm compared to pre-treatment levels. In control L-NAME rats, MAP increased by 31±4 mmHg, while HR was not altered significantly, compared to pre-treatment levels. Chronic leptin infusion alone did not alter glomerular filtration rate (GFR) or renal plasma flow (RPF). However, Leptin+L-NAME reduced GFR by 47±8% and RPF 58±7%; L-NAME alone decreased GFR by 36±11% and RPF 36±17%. Thus, NO does not appear to modulate the anorectic effect of leptin. However, impaired NO synthesis enhances the chronic renal hemodynamic and hypertensive effects of leptin, and greatly amplifies the tachycardia caused by hyperleptinemia, at levels comparable to those found in obesity hypertension.

Role of Nitric Oxide in Modulating the Chronic Cardiovascular and Renal Actions of Leptin

64 Acute studies suggest that leptin has pressor and depressor actions, including stimulation of sympathetic activity as well as increased release of nitric oxide (NO)from the vascular endothelium. The goal of this study was to examine the role of NO in modulating the chronic blood pressure, heart rate, and renal responses to hyperleptinemia, comparable to that found in obesity hypertension. Male Sprague-Dawley rats were implanted with arterial and venous catheters and mean arterial pressure (MAP) and heart rate (HR) were monitored continuously 24h/d. After a 4 day control period, rats were treated with vehicle (n=6) or L-NAME (n=7) for 7 days. After 7 days of vehicle or L-NAME (10 μg/kg/min) treatment to inhibit NO synthesis, rats received a low rate (0.5 μg/kg/min) leptin infusion for 7 days followed by a moderate rate (1.0 μg/kg/min) leptin infusion for 7 days, along with vehicle or L-NAME. A 21 day infusion of L-NAME alone (n=6) served as a control for the leptin+L-NAME rats. The low and moderate leptin infusions reduced food intake by 53±2%and 51±7%, respectively, but L-NAME did not alter the anorectic effect of leptin. Although the low dose of leptin alone did not significantly elevate MAP, it raised HR by 18±3 bpm. The moderate dose of leptin raised MAP from 96±3 to 104±3, but did not increase HR further. In rats pre-treated with L-NAME, the moderate dose of leptin raised MAP by 40±5 mmHg and HR by 73±17 bpm compared to pre-treatment levels. In control L-NAME rats, MAP increased by 31±4 mmHg, while HR was not altered significantly, compared to pre-treatment levels. Chronic leptin infusion alone did not alter glomerular filtration rate (GFR) or renal plasma flow (RPF). However, Leptin+L-NAME reduced GFR by 47±8% and RPF 58±7%; L-NAME alone decreased GFR by 36±11% and RPF 36±17%. Thus, NO does not appear to modulate the anorectic effect of leptin. However, impaired NO synthesis enhances the chronic renal hemodynamic and hypertensive effects of leptin, and greatly amplifies the tachycardia caused by hyperleptinemia, at levels comparable to those found in obesity hypertension.

Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly.

The crucial role of glucocorticoids in obesity and insulin resistance and the actions of the OB protein leptin on the hypothalamic-pituitary-adrenal (HPA) axis suggest that there is an important interaction of leptin with the glucocorticoid system. Therefore, we designed a study to test the effect of leptin directly on adrenocortical steroidogenesis. Primary cultures of bovine adrenocortical cells were incubated with increasing concentrations (10-1,000 ng/ml) of recombinant mouse leptin for 24 h, and the effects of leptin on basal and ACTH-stimulated cortisol secretion were determined. The accumulation of P450 17alpha mRNA following incubation with ACTH (10 nmol/l) and leptin (10-1,000 ng/ml) was analyzed by Northern blot. Adrenocortical cells were characterized by immunohistochemical staining for 17alpha-hydroxyprogesterone. Leptin (10-1,000 ng/ml) inhibited basal and ACTH-stimulated cortisol release. At a concentration that occurs in obese individuals in vivo (100 ng/ml), it reduced basal cortisol secretion to 52.7 +/- 37% (mean +/- SE). The rise in cortisol secretion following maximal ACTH stimulation (10 nmol/l) was blunted to 55.2 +/- 27%. At more physiological concentrations of ACTH (0.1 nmol/l), the inhibition of cortisol release by coincubation with low doses of leptin (10 ng/ml) was even more pronounced, leading to a reduction to 32.8% (1,248 +/- 134 vs. 410 +/- 157 nmol/l). Addition of OB protein (10-1,000 ng/ml) led to a dose-dependent reduction of ACTH-stimulated cytochrome P450 17alpha mRNA accumulation (from 80 to 45%), suggesting that leptin regulates adrenal steroidogenesis at the transcriptional level. These data clearly demonstrate that leptin inhibits cortisol production in adrenocortical cells and therefore appears to be a metabolic signal that directly acts on the adrenal gland.

Chronic Stimulation of Leptin on Food Intake and Body Weight after Microinjection into the Ventromedial Hypothalamus of Conscious Rats

A very low dose of leptin (50 ng) was microinjected into the ventro-medial hypothalamus (VMH) of each rat daily once for three days. Food intake and body weight were measured after leptin injections. Microinjection of leptin into the VMH reduced food intake by 33.3 % significantly (P&lt;0.01) during three days of leptin injection compared to the control. Body weight was measured after 24 h, 48 h and 72 h of leptin injection. After 24 h (P&lt;0.01) and 48 h (P&lt;0.05) of leptin injection, body weight was reduced significantly compared to that of rat before injection. Similarly, after 72 h of leptin injection, a significant reduced body weight was observed (P&lt;0.1). A significant (P&lt;0.001) reduced changes of body weight were found after 24 h, 48 h and 72 h after injection into the VMH when compared to the respective controls injected with saline. The results suggest that leptin has dramatic effect on reducing body weight by inhibition of food intake. doi: 10.3329/taj.v19i2.3154 TAJ 2006; 19(2): 70-75