Co-administration Of Leptin Research Articles

Perinatal Treatment with Leptin, but Not Celastrol, Protects from Metabolically Obese, Normal-Weight Phenotype in Rats

Perinatal nutrition has a well-known influence on obesity susceptibility. We previously demonstrated the protective anti-obesity effects of perinatal leptin administration. Celastrol is a natural compound acting as a leptin sensitizer with anti-obesity effects when administered in adult animals. Here, we aimed to determine if perinatal treatment with leptin, celastrol, or their combination was able to improve metabolic health in animals fed an isocaloric high-fat (HF) diet. Leptin and/or celastrol or their vehicle were administered orally to rats during the suckling period. After weaning, animals were chronically pair-fed with an HF diet provided isocaloric to the intake of a normal-fat diet by control animals to avoid obesity. Isocaloric HF feeding in vehicle-treated animals resulted in metabolic features characteristic of the metabolically obese, normal-weight (MONW) phenotype, i.e., obesity-related disturbances without increased body weight. Leptin treatment prevented liver fat deposition and insulin resistance, induced greater insulin and leptin signaling capacity, decreased gene expression of orexigenic signals at the hypothalamic level, and induced browning in retroperitoneal adipose tissue. However, celastrol treatment did not provide any protective effect and resulted in greater size of the retroperitoneal adipose depot, higher circulating glucose and insulin levels, and decreased leptin sensitivity capacity in adipose tissue. The co-administration of leptin ameliorated the negative effects of celastrol on the retroperitoneal depot, inducing browning and decreasing its size. In conclusion, the perinatal administration of leptin, but not celastrol, provided protection against the consequences of dietary unbalances leading to an MONW phenotype in adulthood.

Ghrelin signaling contributes to fasting-induced attenuation of hindbrain neural activation and hypophagic responses to systemic cholecystokinin in rats.

In rats, overnight fasting reduces the ability of systemic cholecystokinin-8 (CCK) to suppress food intake and to activate cFos in the caudal nucleus of the solitary tract (cNTS), specifically within glucagon-like peptide-1 (GLP-1) and noradrenergic (NA) neurons of the A2 cell group. Systemic CCK increases vagal sensory signaling to the cNTS, an effect that is amplified by leptin and reduced by ghrelin. Since fasting reduces plasma leptin and increases plasma ghrelin levels, we hypothesized that peripheral leptin administration and/or antagonism of ghrelin receptors in fasted rats would rescue the ability of CCK to activate GLP-1 neurons and a caudal subset of A2 neurons that coexpress prolactin-releasing peptide (PrRP). To test this, cFos expression was examined in ad libitum-fed and overnight food-deprived (DEP) rats after intraperitoneal CCK, after coadministration of leptin and CCK, or after intraperitoneal injection of a ghrelin receptor antagonist (GRA) before CCK. In fed rats, CCK activated cFos in ~60% of GLP-1 and PrRP neurons. Few or no GLP-1 or PrRP neurons expressed cFos in DEP rats treated with CCK alone, CCK combined with leptin, or GRA alone. However, GRA pretreatment increased the ability of CCK to activate GLP-1 and PrRP neurons and also enhanced the hypophagic effect of CCK in DEP rats. Considered together, these new findings suggest that reduced behavioral sensitivity to CCK in fasted rats is at least partially due to ghrelin-mediated suppression of hindbrain GLP-1 and PrRP neural responsiveness to CCK.

Interaction between leptin and glutamatergic system on food intake regulation in neonatal chicken: role of NMDA and AMPA receptors

Objective: The aim of the current study was to determine the possible interaction of the central leptin and Glutamatergic systems on feeding behavior in neonatal 3-hours food deprived (FD3) broilers chickens.Methods: In experiment 1, FD3 chicken received intracerebroventricular (ICV) injection of control solution (group i) and 2.5, 5 and 10 µg of Leptin (groups ii–iv). In experiment 2, FD3 chicken were ICV injected with (group i) control solution and groups ii–iv with 2.5, 5 and 10 nmol of AG-490 (JAK2 antagonist). In experiment 3, injections were (i) control solution, (ii) Leptin (10 µg), (iii) AG-490 (2.5 nmol) and (iv) Leptin + AG-490. In experiment 4, broiler chickens were ICV injected with (i) control solution, (ii) Leptin (10 µg), (iii) MK-801 (NMDA glutamate receptors antagonist; 15 nmol) and (iv) Leptin + MK-801. Experiments 5–9 were similar to experiment 1, except chicken were ICV injected with CNQX (AMPA receptor antagonist, 390 nmol), UBP-302 (Kainate receptor antagonist, 390 nmol), AIDA (mGluR1 antagonist, 2 nmol), LY341495 (mGluR2 antagonist, 150 nmol) and UBP1112 (mGluR3 antagonist, 2 nmol) instead of MK-801. Then, food intake was measured until 120 min after injection.Results: ICV injection of leptin (2.5, 5 and 10 µg) significantly decreased food intake in a dose dependent manner (p < 0.05). Also, ICV injection of the JAK2 antagonist (2.5, 5 and 10 nmol) had hyperphagic effect in chicken (p < 0.05). Co-administration of leptin + AG-490, partially decreased leptin-induced hypophagia in broiler chicken (p < 0.05). In addition, co-injection of leptin + MK-801 significalty inhibited leptin-induced hypophagia in neonatal chicken (p < 0.05). Also, co-administration of leptin + CNQX partially attenuated hypophagic effect of leptin in chicken (p < 0.05).Conclusion: The results of present study suggest that leptin has hypophagic effect in neonatal chicken and this effect is probably mediated via NMDA and AMPA glutamatergic receptors.

Oxytocin Administration Alleviates Acute but Not Chronic Leptin Resistance of Diet-Induced Obese Mice

Whereas leptin administration only has a negligible effect on the treatment of obesity, it has been demonstrated that its action can be improved by co-administration of leptin and one of its sensitizers. Considering that oxytocin treatment decreases body weight in obese animals and humans, we investigated the effects of oxytocin and leptin cotreatment. First, lean and diet-induced obese (DIO) mice were treated with oxytocin for 2 weeks and we measured the acute leptin response. Second, DIO mice were treated for 2 weeks with saline, oxytocin (50 μg/day), leptin (20 or 40 µg/day) or oxytocin plus leptin. Oxytocin pre-treatment restored a normal acute leptin response, decreasing food intake and body weight gain. Chronic continuous administration of oxytocin or leptin at 40 µg/day decreased body weight in the presence (leptin) or in the absence (oxytocin) of cumulative differences in food intake. Saline or leptin treatment at 20 µg/day had no impact on body weight. Oxytocin and leptin cotreatments had no additional effects compared with single treatments. These results point to the fact that chronic oxytocin treatment improves the acute, but not the chronic leptin response, suggesting that this treatment could be used to improve the short-term satiety effect of leptin.

Methylnaltrexone potentiates body weight and fat reduction with leptin

Leptin increases energy expenditure by enhancing systemic and brown adipose metabolism. In a neonatal rat model, retroperitoneal fat pad weight decreased significantly in leptin-treated animals, which reduced body weight. As opioids increase feeding, opioid antagonists may decrease food intake and body weight. However, interactions between leptin and the activity of peripheral opioids on body weight and fat accumulation have not been investigated. In this study, the authors evaluated the effects of naloxone (a nonselective opioid antagonist) and methylnaltrexone (a peripherally acting opioid antagonist) on the action of leptin in neonatal rats. Compared with control, the weight gain of pups given a single daily intraperitoneal injection of leptin 0.5 mg/kg, leptin 0.5 mg/kg plus naloxone 0.3 mg/kg, or leptin 0.5 mg/kg plus methylnaltrexone 3.0 mg/kg for 8 consecutive days was significantly reduced (all p < 0.01). Naloxone or methylnaltrexone significantly potentiated leptin's effect on body weight (p < 0.05 or p < 0.01, respectively). After coadministration of leptin plus naloxone or leptin plus methylnaltrexone, weight reduction in the right retroperitoneal fat pads was also significant compared with the reduction after leptin alone (p < 0.05 or p < 0.01, respectively). The data suggest the existence of a peripheral opioid-related mechanism in leptin-active modulation of body weight.

Synthesis of New 7-bromo-5-(2’-chlorophenyl)-3-arylamino-1,2-dihydro-3H-1,4-benzodiazepine Derivatives and Their Influence on Appetite in Rats

A series of new 7-bromo-5-(2′-chlorophenyl)-3-arylamino-1,2-dihydro-3H-1,4-benzodiazepine derivatives (II-VIII) were synthesized. The series of synthesized compounds included those that had a substantial effect on appetite in rats, exhibiting both orexigenic and anorexigenic effects at low doses (2.07 – 2.21 μM). Compound VI had anorexigenic activity comparable with that of the hormone leptin, which reduces the appetite and food intake. Co-administration of leptin and III, which exhibited a pronounced orexigenic effect, reduced appetite by 73%.

Leptin Administration Enhances Islet Transplant Performance in Diabetic Mice

Islet transplantation is an effective method to obtain long-term glycemic control for patients with type 1 diabetes, yet its widespread use is limited by an inadequate supply of donor islets. The hormone leptin has profound glucose-lowering and insulin-sensitizing action in type 1 diabetic rodent models. We hypothesized that leptin administration could reduce the dose of transplanted islets required to achieve metabolic control in a mouse model of type 1 diabetes. We first performed a leptin dose-response study in C57Bl/6 mice with streptozotocin (STZ)-induced diabetes to determine a leptin dose insufficient to reverse hyperglycemia. Subsequently, we compared the ability of suboptimal islet transplants of 50 or 125 syngeneic islets to achieve glycemic control in STZ-induced diabetic C57Bl/6 mice treated with or without this dose of leptin. The dose-response study revealed that leptin reverses STZ-induced diabetes in a dose-dependent manner. Supraphysiological leptin levels were necessary to restore euglycemia but simultaneously increased risk of hypoglycemia, and also lost efficacy after 12 days of administration. In contrast, 1 µg/day leptin only modestly reduced blood glucose but maintained efficacy throughout the study duration. We then administered 1 µg/day leptin to diabetic mice that underwent transplantation of 50 or 125 islets. Although these islet doses were insufficient to ameliorate hyperglycemia alone, coadministration of leptin with islet transplantation robustly improved control of glucose and lipid metabolism, without increasing circulating insulin levels. This study reveals that low-dose leptin administration can reduce the number of transplanted islets required to achieve metabolic control in STZ-induced diabetic mice.

Firing Up Brown Fat with Brain Amylin

The search for valuable antiobesity therapies is an urgent necessitynotonly indevelopedbutalsoindevelopingcountries (1). To date, bariatric surgery is the most effective strategy to reduce and maintain weight loss in obese individuals, and therefore much effort is being made to depict which gastrointestinal (GI) signals are involved in this process and their mechanisms of action (2). Among the large list of GI factors controlling energy balance, amylin is one of the most attractive. Amylin is a 37-amino acid peptide hormone that is cosecreted with insulin by pancreatic -cells in response to nutrient ingestion, helping to maintain glucose homeostasis (3). Amylin has been involved in a wide range of biological actions, including slow gastric emptying (4), suppression of nutrient-stimulated glucagon secretion (5), and suppression of food intake (6). Importantly, amylin maintains its antiobesity action in obese rats (7) and is able to restore leptin sensitivity in obese rodents and humans (8). In addition, combined amylin and leptin treatment has synergistic actions in obese animals (9), an interaction that does not occur with other GI signals such as glucagon like peptide-1 (10). The body weight loss caused by the coadministration of leptin and amylin cannot be entirely explained by the suppression in food intake (7), suggesting that alternative metabolic pathways are involved in amylin’s control of body weight. Those food intake–independent metabolic actions seem to be, at least partially, mediated by the brain, because chronic central infusion of amylin reduced the respiratory exchange ratio and increased body temperature (11). Mice overexpressing neuronal RAMP1 (receptor activity–modifying protein 1, the amylin receptor) also show higher energy expenditure as indicated by increased oxygen consumption, body temperature, sympathetic tone, and expression of thermogenic markers in brown adipose tissue (BAT) (12). In this issue of Endocrinology, Rahmouni and colleagues (13) move our neurobiological understanding significantly forwardbydemonstratingadirect linkbetweenbrainamylin and BAT thermogenesis through the sympathetic nervous system (SNS). Specifically they show that besides its role on feeding, amylin increases the activity of the SNS, leading to increasedBATthermogenesis andweight loss.Theyusemice and direct multifiber sympathetic nerve recording to unequivocally demonstrate that intracerebroventricular injection of amylin promotes a dose-dependent increase in the sympathetic nerve activity subserving BAT, which subsequently elevates body temperature. The connotation of these data is exemplified by several mechanistic findings, which demonstrate the crucial effect of the amylin-SNS-BAT axis. First, intracerebroventricular pretreatment with the amylin receptor antagonist AC187 totally blunted the BAT SNA response induced by central amylin (13). Second, the amylin response was boosted in transgenic mice overexpressing the amylin receptor subunit RAMP1 in the central nervous system (13). Finally, central amylin promoted a marked activation (measured as c-Fos immunoreactivity) in key hypothalamic and brainstem sites such as the anterior hypothalamic area (AHA), the dorsomedial nucleus of the hypothalamus (DMH), the area postrema, and the nucleus of the tractus solitarius, all of which are known to modulate energy balance (14). This specific pattern of activation also opens up some interesting questions about the neuronal, cellular, and molecular mechanisms mediating amylin’s action. Although a role for the AHA and the DMH in regulating BAT has been known since the 1980s (15), only recently have reports started to address the molecular mech-

Amylin-induced downregulation of hippocampal neurogenesis is attenuated by leptin in a STAT3/AMPK/ERK-dependent manner in mice

Both leptin and insulin sensitivity have been linked with pathophysiological processes involving the central nervous system in general, and the hippocampus in particular, but the role of leptin in hippocampal neurogenesis has not yet been elucidated. Also, no previous studies have evaluated whether amylin or the endogenous insulin sensitiser adiponectin interact with leptin to alter hippocampal neurogenesis in mouse hippocampal neuronal (HN) cells or investigated the role of leptin, amylin or adiponectin signalling in mouse HN cells. Hippocampal neurogenesis and leptin, amylin and adiponectin signalling were studied in vitro using mouse H19-7 HN cell lines. Amylin decreased cell proliferation in a dose-dependent manner. This effect was diminished by leptin administration and was dependent on signal transducer and activator of transcription 3 (STAT3)/AMP-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK). Adiponectin effects were null. We also observed, using immunocytochemical analysis, that amylin decreased activation of microtubule-associated protein 2, a specific neurite outgrowth marker, and synapsin, a specific synaptogenesis marker. By contrast, both effects were attenuated by co-administration of leptin. Finally, we observed that these effects were blocked by pre-treatment with AG490, a STAT3 inhibitor, and STAT3 small interfering RNA administration. Our data suggest that amylin in pharmacological concentrations may have a neurotoxic effect whereas leptin in physiological and pharmacological concentrations has a protective effect counteracting amylin-decreased hippocampal neurogenesis via STAT3/AMPK/ERK signalling in mouse H19-7 HN cell lines. Overall, our data support a novel role for leptin and amylin in the processes of mouse hippocampal neurogenesis and provide new insights into the mechanisms of neurogenic regulation.

Restoration of leptin responsiveness in diet‐induced obese mice using an optimized leptin analog in combination with exendin‐4 or FGF21

The identification of leptin as a mediator of body weight regulation provided much initial excitement for the treatment of obesity. Unfortunately, leptin monotherapy is insufficient in reversing obesity in rodents or humans. Recent findings suggest that amylin is able to restore leptin sensitivity and when used in combination with leptin enhances body weight loss in obese rodents and humans. However, as the uniqueness of this combination therapy remains unclear, we assessed whether co-administration of leptin with other weight loss-inducing hormones equally restores leptin responsiveness in diet-induced obese (DIO) mice. Accordingly, we report here the design and characterization of a series of site-specifically enhanced leptin analogs of high potency and sustained action that, when administered in combination with exendin-4 or fibroblast growth factor 21 (FGF21), restores leptin responsiveness in DIO mice after an initial body weight loss of 30%. Using either combination, body weight loss was enhanced compared with either exendin-4 or FGF21 monotherapy, and leptin alone was sufficient to maintain the reduced body weight. In contrast, leptin monotherapy proved ineffective when identical weight loss was induced by caloric restriction alone over a comparable time. Accordingly, we find that a hypothalamic counter-regulatory response to weight loss, assessed using changes in hypothalamic agouti related peptide (AgRP) levels, is triggered by caloric restriction, but blunted by treatment with exendin-4. We conclude that leptin re-sensitization requires pharmacotherapy but does not appear to be restricted to a unique signaling pathway. Our findings provide preclinical evidence that high activity, long-acting leptin analogs are additively efficacious when used in combination with other weight-lowering agents.

Effects of leptin replacement alone and with exendin-4 on food intake and weight regain in weight-reduced diet-induced obese rats

Weight loss in obese humans produces a relative leptin deficiency, which is postulated to activate potent orexigenic and energy conservation mechanisms to restrict weight loss and promote weight regain. Here we determined whether leptin replacement alone or with GLP-1 receptor agonist exendin-4 attenuates weight regain or promotes greater weight loss in weight-reduced diet-induced obese (DIO) rats. Forty percent restriction in daily intake of a high-fat diet in DIO rats for 4 wk reduced body weight by 12%, body fat by 29%, and plasma leptin by 67% and normalized leptin sensitivity. When food restriction ended, body weight, body fat, and plasma leptin increased rapidly. Daily administration of leptin [3-h intraperitoneal (ip) infusions (4 nmol·kg(-1)·h(-1))] at onset and end of dark period for 3 wk did not attenuate hyperphagia and weight regain, nor did it affect mean daily meal sizes or meal numbers. Exendin-4 (50 pmol·kg(-1)·h(-1)) infusions during the same intervals prevented postrestriction hyperphagia and weight regain by normalizing meal size. Coadministration of leptin and exendin-4 did not reduce body weight more than exendin-4 alone. Instead, leptin began to attenuate the inhibitory effects of exendin-4 on food intake, meal size, and weight regain by the end of the second week of administration. Plasma leptin in rats receiving leptin was sevenfold greater than in rats receiving vehicle and 17-fold greater than in rats receiving exendin-4. Together, these results do not support the hypothesis that leptin replacement alone or with exendin-4 attenuates weight regain or promotes greater weight loss in weight-reduced DIO rats.

Leptin Selectively Augments Thymopoiesis in Leptin Deficiency and Lipopolysaccharide-Induced Thymic Atrophy

The thymus is a lymphoid organ that selects T cells for release to the peripheral immune system. Unfortunately, thymopoiesis is highly susceptible to damage by physiologic stressors and can contribute to immune deficiencies that occur in a variety of clinical settings. No treatment is currently available to protect the thymus from stress-induced involution. Leptin-deficient (ob/ob) mice have severe thymic atrophy and this finding suggests that this hormone is required for normal thymopoiesis. In this study, the ability of leptin to promote thymopoiesis in wild-type C57BL/6 and BALB/c mice, as well as in leptin-deficient (ob/ob) and endotoxin-stressed (Escherichia coli LPS) mice, was determined. Leptin administration induced weight loss and stimulated thymopoiesis in ob/ob mice, but did not stimulate thymopoiesis in wild-type C57BL/6 nor BALB/c mice. In endotoxin-stressed mice, however, leptin prevented LPS-induced thymus weight loss and stimulated TCRalpha gene rearrangement. Coadministration of leptin with LPS blunted endotoxin-induced systemic corticosterone response and production of proinflammatory cytokines. Thus, leptin has a selective thymostimulatory role in settings of leptin deficiency and endotoxin administration, and may be useful for protecting the thymus from damage and augmenting T cell reconstitution in these clinical states.

Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibers

Leptin and cholecystokinin (CCK) have a synergistic interaction in the suppression of food intake, and afford similar gastroprotective activity. The present study was designed to investigate the putative protective effects of CCK and leptin on acute colonic inflammation. Leptin or CCK-8s was injected to rats intraperitoneally immediately before and 6 h after the induction of colitis with acetic acid. CCK-A receptor antagonist (L-364,718) or CCK-B receptor antagonist (L-365,260) was injected intraperitoneally 15 min before leptin or CCK treatments. In a group of rats, vagal afferent fibers were denervated by topical application of capsaicin on the cervical vagi. Rats were decapitated at 24 h, and the distal 8 cm of the colon were removed for macroscopic scoring, determination of tissue wet weight index (WWI), histologic assessment and tissue myeloperoxidase (MPO) activity. All inflammation parameters were increased by acetic acid-induced colitis compared to control group. Leptin or CCK-8s treatment reduced these parameters in a similar manner, while co-administration of leptin and CCK was found to be more effective in reducing the macroscopic score and WWI. CCK-8s-induced reduction in the score and WWI was prevented by CCK-A, but not by CCK-B receptor antagonist, whereas neither antagonist altered the inhibitory effect of leptin on colitis-induced injury. On the other hand, perivagal capsaicin prevented the protective effects of both CCK-8s and leptin on colitis. Our results indicate that leptin and CCK have anti-inflammatory effects on acetic acid-induced colitis in rats, which appear to be mediated by capsaicin-sensitive vagal afferent fibers involving the reduction in colonic neutrophil infiltration.

Leptin as an adjunct of insulin therapy in insulin-deficient diabetes.

The purpose of this study was to assess the therapeutic implication of leptin in insulin-deficient diabetes. Insulin-deficient diabetes was induced by streptozotocin (STZ) in transgenic skinny mice overexpressing leptin. Plasma concentrations of glucose, insulin, and leptin were measured. The effects on body weight, food intake, and hypothalamic gene expressions were analyzed. After diabetes was induced, graded doses of insulin ranging from 0.4 to 51.2 mU.g(-1).day(-1) were injected. Co-administration of leptin and insulin was also carried out using osmotic pumps. After STZ injection, both transgenic and non-transgenic littermates developed marked hyperglycaemia as a result of severe hypoinsulinaemia [termed diabetic transgenic skinny mice overexpressing leptin (diabetic TGM) and diabetic non-transgenic littermates (diabetic WT) respectively], although diabetic TGM were more sensitive to exogenously administered insulin than diabetic WT. Diabetic WT were hypoleptinaemic and hyperphagic relative to non-diabetic WT, whereas diabetic TGM, which remained hyperleptinaemic, were less hyperphagic than diabetic WT. After STZ injection, hypothalamic expressions of orexigenic and anorexigenic peptide mRNAs were up-regulated and down-regulated, respectively, in diabetic WT, whereas they were unchanged in diabetic TGM. Diabetic TGM became normoglycaemic, when treated with insulin at such doses that did not improve hyperglycaemia in diabetic WT. We found that a sub-threshold dose of insulin that does not affect glucose homeostasis is effective in improving the diabetes in normal mice rendered diabetic by STZ injection, when combined with leptin. This study suggests that leptin could be used as an adjunct of insulin therapy in insulin-deficient diabetes, thereby providing an insight into the therapeutic implication of leptin as an anti-diabetic agent.

Effects of leptin on endothelial function with OB-Rb gene transfer in Zucker fatty rats

The metabolic syndrome in association with obesity is a major clinical problem inducing hypertension, diabetes mellitus, and atherosclerosis. Leptin induces angiogenesis by its proliferative effects on endothelial cells (ECs) via OB receptor (OB-Rb) gene. We evaluated the growth of ECs and intracellular signalings in response to leptin in vitro and the angiogenic effects of leptin in the cornea in vivo with and without adenovirus-mediated transfer of the OB-Rb gene in Zucker fatty (ZF) rats as a model for the metabolic syndrome. Recombinant adenovirus vector encoding rat OB-Rb (Ad.OB-Rb) or Escherichia coli. LacZ (Ad.Lac Z) was transfected into cultured ECs from Zucker lean (ZL) rats and ZF rats. Leptin increased DNA synthesis dose-dependently in ECs from ZL rats but not ZF rats. Infection with Ad.OB-Rb, but not with Ad.LacZ, improved the growth effects of leptin in ECs from ZF rats. Leptin induced phosphorylation of Janus kinase (JAK)2, signal transducer and activator of transcription (STAT)3, and extracellular signal-regulated kinase (ERK) in ECs from ZL rats but not ZF rats. Infection with Ad.OB-Rb restored phosphorylation of JAK2 and STAT3 in ECs from ZF rats. Leptin induced angiogenesis in cornea from ZL rats, but not from ZF rats. Coadministration of leptin and Ad.OB-Rb induced angiogenesis in cornea from ZF rats. Ad.LacZ did not influence the angiogenic effects of leptin. The impaired endothelial function with the leptin resistance may be one of causes of the atherosclerosis in the metabolic syndrome.

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.