Intracerebroventricular Leptin Administration Research Articles

Understanding leptin-dependent regulation of skeletal homeostasis

Despite growing evidence for adipose tissue regulation of bone mass, the role of the adipokine leptin in bone remodeling remains controversial. The majority of in vitro studies suggest leptin enhances osteoblastic proliferation and differentiation while inhibiting adipogenic differentiation from marrow stromal cells. Alternatively, some evidence demonstrates either no effect or a pro-apoptotic action of leptin on stromal cells. Similarly, in vivo work has demonstrated both positive and negative effects of leptin on bone mass. Most of the literature supports the idea that leptin suppresses bone mass by acting in the brainstem to reduce serotonin-dependent sympathetic signaling from the ventromedial hypothalamus to bone. However, other studies have found partly or entirely contrasting actions of leptin. Recently one study found a significant effect of surgery alone with intracerebroventricular administration of leptin, a technique crucial for understanding centrally-mediated leptin regulation of bone. Thus, two mainstream hypotheses for the role of leptin on bone emerge: 1) direct regulation through increased osteoblast proliferation and differentiation and 2) indirect suppression of bone formation through a hypothalamic relay. At the present time, it remains unclear whether these effects are relevant in only extreme circumstances (i.e. models with complete deficiency) or play an important homeostatic role in the regulation of peak bone acquisition and skeletal remodeling. Ultimately, determining the actions of leptin on the skeleton will be critical for understanding how the obesity epidemic may be impacting the prevalence of osteoporosis.

The nitric oxide pathway participates in lordosis behavior induced by central administration of leptin

Intracerebroventricular (icv) administration of leptin facilitates lordosis behavior in ad libitum-fed, estrogen-primed rats. The cellular mechanism involved in this response is unknown. The present study tested the hypothesis that the nitric oxide-guanylyl cyclase, cGMP-dependent protein kinase (PKG) pathway is involved in the facilitation of lordosis behavior induced by the central administration of leptin. We tested the importance of the nitric oxide/cGMP pathway for lordosis stimulation by either icv infusion of a nitric oxide synthase inhibitor (L-NAME) or a nitric oxide-dependent, soluble guanylyl cyclase inhibitor (ODQ) 30 min before leptin administration (1 μg). This dose of leptin reliably induced lordosis behavior in ovariectomized estradiol benzoate treated rats. The lordosis induced by leptin at 1 and 2h after infusion was significantly reduced by the previous injection of either L-NAME or by ODQ. Intracerebroventricular infusion of the PKG inhibitor (KT5823) 30 min before leptin infusion, also significantly inhibited the lordosis behavior induced by leptin at 1 and 2h after hormone administration. These data support the hypothesis that the nitric oxide/cGMP/PKG pathway is involved in the facilitation of lordosis by leptin in estrogen-primed female rats.

Chronic Central Leptin Decreases Food Intake and Improves Glucose Tolerance in Diet-Induced Obese Mice Independent of Hypothalamic Malonyl CoA Levels and Skeletal Muscle Insulin Sensitivity

Although acute leptin administration in the hypothalamus decreases food intake and increases peripheral energy metabolism, the peripheral actions of central chronic leptin administration are less understood. In this study, we investigated what effects chronic (7 d) intracerebroventricular (ICV) administration of leptin has on energy metabolism and insulin sensitivity in diet-induced obese mice. C57/BL mice were fed a low-fat diet (LFD; 10% total calories) or high-fat diet (HFD; 60% total calories) for 8 wk after which leptin was administered ICV for 7 consecutive days. Mice fed a HFD showed signs of insulin resistance, as evidenced by an impaired glucose tolerance test. Chronic leptin treatment resulted in a decrease in food intake and body weight and normalization of glucose clearance but no improvement in insulin sensitivity. Chronic ICV leptin increased hypothalamic signal transducer and activator of transcription-3 and AMP-activated protein kinase phosphorylation but did not change hypothalamic malonyl CoA levels in HFD fed and LFD-fed mice. In the gastrocnemius muscles, the levels of malonyl CoA in both leptin-treated groups were lower than their respective control groups, suggesting an increase in fatty acid oxidation. However, only in the muscles of ICV leptin-treated LFD mice was there a decrease in lipid metabolites including diacylglycerol, triacylglycerol, and ceramide. Our results suggest that chronic ICV leptin decreases food consumption and body weight via a mechanism different from acute ICV leptin administration. Although chronic ICV leptin treatment in HFD mice improves glucose tolerance, this occurs independent of changes in insulin sensitivity in the muscles of HFD mice.

Central (ICV) leptin injection increases bone formation, bone mineral density, muscle mass, serum IGF-1, and the expression of osteogenic genes in leptin-deficient ob/ob mice

Both central and peripheral leptin administrations reduce body weight, food intake, and adiposity in ob/ob mice. In this study we compared effects of intracerebroventricular (ICV) and subcutaneous (SC) administration of leptin on bone metabolism in the appendicular and axial skeleton and adipose tissue gene expression and determined the effects of ICV leptin on bone marrow gene expression in ob/ob mice. In experiment 1, leptin (1.5 or 0.38 µg/d) or control was continuously injected ICV for 12 days. Gene expression analysis of femoral bone marrow stromal cells showed that expression of genes associated with osteogenesis was increased after ICV injection, whereas those associated with osteoclastogenesis, adipogenesis, and adipocyte lipid storage were decreased. In experiment 2, leptin was injected continuously ICV (0.0 or 1.5 µg/d) or SC (0.0 or 10 µg/d) for 12 days. In both experiments, regardless of mode of administration, leptin decreased body weight, food intake, and body fat and increased muscle mass, bone mineral density, bone mineral content, bone area, marrow adipocyte number, and mineral apposition rate. Serum insulin was decreased, whereas serum osteocalcin, insulin-like growth factor 1, osteoprotegerin, pyridinoline, and receptor activator of nuclear factor κB ligand concentrations were increased. In experiment 2, expression of genes in adipose tissue associated with apoptosis, lipid mobilization, insulin sensitivity, and thermogenesis was increased, whereas expression of genes associated with cell differentiation and maturation was decreased regardless of mode of administration. Thus ICV injection of leptin promotes expression of pro-osteogenic factors in bone marrow, leading to enhanced bone formation in ob/ob mice.

Brain Angiotensin Generation Contributes to Renal Sympathetic Nerve Responses to Leptin

Leptin acts in the brain to increase sympathetic nerve activity (SNA) in addition to reducing appetite and body weight. We recently demonstrated that pharmacologic blockade of brain angiotensin 1 receptors (AT1R) selectively attenuated SNA responses to intra-cerebroventricular (ICV) administration of leptin in rats without attenuating SNA responses to the melanocortin agonist MTII. We advanced the concept of a brain leptin-RAS interaction in regulation of SNA. To explore the mechanism, we measured angiotensin converting enzyme (ACE) and AT1aR mRNA in arcuate nucleus, paraventricular nucleus, and subfornical organ (SFO). Leptin increased ACE mRNA but only in the SFO, a site of functional leptin as well as angiotensin receptors. In the present study, we tested the hypothesis that ACE induced generation of brain angiotensin contributes to renal SNA responses to leptin. In anesthetized Sprague-Dawley rats, captopril, 12.5 ug ICV, blocked responses to angiotensin I, but not angiotensin II. Following ICV vehicle, leptin, 10 ug ICV, increased renal SNA by +141±38% (mean±SE). In contrast, following captopril ICV, leptin did not increase renal SNA (−14±17%). Captopril alone did not alter renal SNA. These studies support the concept of a brain RAS-leptin interaction and suggest that this interaction involves leptin-induced generation of brain angiotensin.

Thyroid Transcription Factor-1 Regulates Feeding Behavior via Melanocortin Pathway in the Hypothalamus

OBJECTIVEα-Melanocyte–stimulating hormone (α-MSH) and agouti-related peptide (AgRP) control energy homeostasis by their opposing actions on melanocortin receptors (MC3/4R) in the hypothalamus. We previously reported that thyroid transcription factor-1 (TTF-1) controls feeding behavior in the hypothalamus. This study aims to identify the function of TTF-1 in the transcriptional regulation of AgRP and α-MSH synthesis for the control of feeding behavior.RESEARCH DESIGN AND METHODSTTF-1 activity in AgRP and pro-opiomelanocortin (POMC) transcription was examined using gel-shift and promoter assays and an in vivo model of TTF-1 synthesis inhibition by intracerebroventricular injection of an antisense (AS) oligodeoxynucleotide (ODN). Double immunohistochemistry was performed to colocalize TTF-1 and AgRP or α-MSH in the hypothalamic arcuate nucleus (ARC). To determine whether TTF-1 action on food intake is mediated through MC3/4R, we measured changes in food intake upon intracerebroventricular injection of MC3/4R antagonists (SHU9119 and AgRP) into rat brain preinjected with the AS ODN.RESULTSTTF-1 stimulated AgRP but inhibited POMC transcription by binding to the promoters of these genes. TTF-1 was widely distributed in the hypothalamus, but we identified some cells coexpressing TTF-1 and AgRP or α-MSH in the ARC. In addition, intracerebroventricular administration of leptin decreased TTF-1 expression in the hypothalamus, and AS ODN-induced inhibition of TTF-1 expression decreased food intake and AgRP expression but increased α-MSH expression. Anorexia induced by the AS ODN was attenuated by the administration of MC3/4R antagonists.CONCLUSIONSTTF-1 transcriptionally regulates synthesis of AgRP and α-MSH in the ARC and affects feeding behavior via the melanocortin pathway.

Intracerebroventricular Leptin Infusion Improves Glucose Homeostasis in Lean Type 2 Diabetic MKR Mice via Hepatic Vagal and Non-Vagal Mechanisms

MKR mice, lacking insulin-like growth factor 1 receptor (IGF-1R) signaling in skeletal muscle, are lean yet hyperlipidemic, hyperinsulinemic, and hyperglycemic, with severe insulin resistance and elevated hepatic and skeletal muscle levels of triglycerides. We have previously shown that chronic peripheral administration of the adipokine leptin improves hepatic insulin sensitivity in these mice independently of its effects on food intake. As central leptin signaling has been implicated in the control of peripheral glucose homeostasis, here we examined the ability of central intracerebroventricular leptin administration to affect energy balance and peripheral glucose homeostasis in non-obese diabetic male MKR mice. Central leptin significantly reduced food intake, body weight gain and adiposity, as well as serum glucose, insulin, leptin, free fatty acid and triglyceride levels relative to ACSF treated controls. These reductions were accompanied by increased fat oxidation as measured by indirect calorimetry, as well as increased oxygen consumption. Central leptin also improved glucose tolerance and hepatic insulin sensitivity determined using the euglycemic-hyperinsulinemic clamps relative to pair fed vehicle treated controls, as well as increasing the rate of glucose disappearance. Hepatic vagotomy only partially reversed the ability of central leptin to improve glucose tolerance. These results demonstrate that central leptin dramatically improves insulin sensitivity independently of its effects on food intake, in a lean mouse model of type 2 diabetes. The findings also suggest that: 1) both hepatic vagal and non-vagal pathways contribute to this improvement, and 2) central leptin alters glucose disposal in skeletal muscle in this model.

Intracerebroventricular Leptin Administration Differentially Alters Cardiac Energy Metabolism in Mice Fed a Low-fat and High-fat Diet

Leptin directly acts on peripheral tissues and alters energy metabolism in obese mice. It also has acute beneficial effects on these tissues via its hypothalamic action. However, it is not clear what effect chronic intracerebroventrical (ICV) leptin administration has on cardiac energy metabolism. We examined the effects of chronic ICV leptin on glucose and fatty acid metabolism in isolated working hearts from high-fat-fed and low-fat-fed mice. Mice were fed a high-fat (60% calories from fat) or low-fat (10% calories from fat) diet for 8 weeks before ICV leptin (5 [mu]g/d) for 7 days. In low-fat-fed mice, leptin increased glucose oxidation rates in isolated working hearts when compared with control [203 +/- 21 vs. 793 +/- 93 nmol[middle dot](g dry weight)-1[middle dot]min-1]. In high-fat-fed mice leptin inhibited fatty acid oxidation [476 +/- 73 vs. 251 +/- 38 nmol[middle dot](g[middle dot]dry[middle dot]wt)-1[middle dot]min-1]. The increase in glucose oxidation in low-fat-fed mice was accompanied by increased pyruvate dehydrogenase activity. In high-fat-fed mice, leptin increased cardiac malonyl coenzyme A levels, secondary to a decrease in malonyl coenzyme A decarboxylase expression. These results suggest that ICV leptin alters cardiac energy metabolism opposite to its peripheral effects and that these effects differ depending on energy substrate supply to the mice.

Central infusion of leptin improves insulin resistance and suppresses β-cell function, but not β-cell mass, primarily through the sympathetic nervous system in a type 2 diabetic rat model

Aims We investigated whether hypothalamic leptin alters β-cell function and mass directly via the sympathetic nervous system (SNS) or indirectly as the result of altered insulin resistant states. Main methods The 90% pancreatectomized male Sprague Dawley rats had sympathectomy into the pancreas by applying phenol into the descending aorta (SNSX) or its sham operation (Sham). Each group was divided into two sections, receiving either leptin at 300 ng/kg bw/h or artificial cerebrospinal fluid (aCSF) via intracerebroventricular (ICV) infusion for 3 h as a short-term study. After finishing the infusion study, ICV leptin (3 μg/kg bw/day) or ICV aCSF (control) was infused in rats fed 30 energy % fat diets by osmotic pump for 4 weeks. At the end of the long-term study, glucose-stimulated insulin secretion and islet morphometry were analyzed. Key findings Acute ICV leptin administration in Sham rats, but not in SNSX rats, suppressed the first- and second-phase insulin secretion at hyperglycemic clamp by about 48% compared to the control. Regardless of SNSX, the 4-week administration of ICV leptin improved glucose tolerance during oral glucose tolerance tests and insulin sensitivity at hyperglycemic clamp, compared to the control, while it suppressed second-phase insulin secretion in Sham rats but not in SNSX rats. However, the pancreatic β-cell area and mass were not affected by leptin and SNSX, though ICV leptin decreased individual β-cell size and concomitantly increased β-cell apoptosis in Sham rats. Significance Leptin directly decreases insulin secretion capacity mainly through the activation of SNS without modulating pancreatic β-cell mass.

Exposure to a High-Fat Diet Alters Leptin Sensitivity and Elevates Renal Sympathetic Nerve Activity and Arterial Pressure in Rabbits

The activation of the sympathetic nervous system through the central actions of the adipokine leptin has been suggested as a major mechanism by which obesity contributes to the development of hypertension. However, direct evidence for elevated sympathetic activity in obesity has been limited to muscle. The present study examined the renal sympathetic nerve activity and cardiovascular effects of a high-fat diet (HFD), as well as the changes in the sensitivity to intracerebroventricular leptin. New Zealand white rabbits fed a 13.5% HFD for 4 weeks showed modest weight gain but a 2- to 3-fold greater accumulation of visceral fat compared with control rabbits. Mean arterial pressure, heart rate, and plasma norepinephrine concentration increased by 8%, 26%, and 87%, respectively (P<0.05), after 3 weeks of HFD. Renal sympathetic nerve activity was 48% higher (P<0.05) in HFD compared with control diet rabbits and was correlated to plasma leptin (r=0.87; P<0.01). Intracerebroventricular leptin administration (5 to 100 microg) increased mean arterial pressure similarly in both groups, but renal sympathetic nerve activity increased more in HFD-fed rabbits. By contrast, intracerebroventricular leptin produced less neurons expressing c-Fos in HFD compared with control rabbits in regions important for appetite and sympathetic actions of leptin (arcuate: -54%, paraventricular: -69%, and dorsomedial hypothalamus: -65%). These results suggest that visceral fat accumulation through consumption of a HFD leads to marked sympathetic activation, which is related to increased responsiveness to central sympathoexcitatory effects of leptin. The paradoxical reduction in hypothalamic neuronal activation by leptin suggests a marked "selective leptin resistance" in these animals.

Regional and temporal differences in leptin signaling in rat brain

Leptin regulates energy homeostasis through activation of different hypothalamic pathways. Evidence indicates that leptin is a pleiotropic hormone that acts on many brain areas, altering food intake, metabolism, and locomotion, among other functions. Because short-term effects of leptin infusion and intracellular pathways in other brain areas involved in food regulation have not been thoroughly analysed, we have studied the acute effect of intracerebroventricular leptin administration on the levels of the long form of leptin receptor (Ob-Rb), as well as on activation of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3), protein kinase B (Akt), extracellular regulated kinases (ERKs) and levels of suppressor of cytokine signaling-3 (SOCS3) in the hypothalamus, hippocampus, frontal cortex and cerebellum of adult male Wistar rats at 15 min, 1 and 6 h. The levels of Ob-Rb increased at 6 h in hypothalamus only. Leptin activated the JAK2/STAT3 pathway in all areas, although in a temporally specific pattern. In contrast, this hormone decreased Akt activation in hypothalamus, hippocampus and cerebellum and ERK activation in frontal cortex, while it increased ERK activation in hypothalamus and hippocampus. These differences in modulation of Ob-Rb levels and signaling indicate that the rapid effects of leptin in non-hypothalamic areas are mediated, at least in part, through the intracellular pathways involved in hypothalamic energy balance, but in a temporally specific manner.

Up-regulation of apolipoprotein E by leptin in the hypothalamus of mice and rats

Apolipoprotein E (apoE) is a satiation factor, playing an important role in the regulation of food intake and body weight. We previously reported that apoE was present in the hypothalamus, but it is unclear which type of the cells in this brain area expressing apoE. In addition, hypothalamic apoE mRNA levels were significantly reduced in both genetically obese ob/ob (leptin deficient) mice and high-fat diet-induced obese (leptin resistant) rats, raising the possibility that deficient leptin signaling might be related to the change in apoE gene expression. In the present studies, using double-staining immunohistochemistry, we demonstrated that apoE is mainly present in astrocytes. To characterize the effect of leptin on apoE gene expression, ob/ ob and db/db mice were treated with recombinant mouse leptin (3 μg/g daily, i.p.) or vehicle for 5 days. We found that the increased hypothalamic apoE mRNA levels occurred only in leptin-treated ob/ob, but not in pair-fed ob/ob, or db/db, mice, indicating that leptin up-regulated hypothalamic apoE gene expression depends upon an intact leptin receptor, and this effect is not related to the changes in food intake and body weight. The reduced apoE gene expression caused by fasting, which also results in relatively lower leptin level, is restored by intracerebroventricular administration of leptin. In addition, leptin was significantly less efficacious in apoE KO mice because these animals consumed more food and lost less weight following leptin treatment, compared with wild-type controls. These observations imply that apoE signaling, at least partially, mediates the inhibitory effects of leptin on feeding.

The action of leptin in the ventral tegmental area to decrease food intake is dependent on Jak-2 signaling.

Recent evidence suggests that leptin reduces food intake via actions in the brain circuitry of food reward, such as the ventral tegmental area (VTA), as leptin receptors are present in the VTA, and leptin injection in the VTA reduces food intake. In the hypothalamus, leptin-induced anorexia requires signaling via Janus kinase-signal transducer and activator of transcription (Jak-STAT), insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI 3-kinase), and mammalian target of rapamycin (mTOR). In this study, we determined whether leptin activates each of these signal transduction pathways in the VTA and whether these signaling pathways are required for VTA-leptin induced anorexia. Here, we show that pSTAT3-Tyr(705), a marker of leptin activation, was induced in a midbrain region containing the VTA and substantia nigra following either intracerebroventricular leptin or direct administration of leptin to the VTA, but these interventions failed to increase levels of either pAKT-Ser(473) or phospho-p70S6K-Thr(389), markers of IRS-PI 3-kinase and mTOR signaling, respectively. Moreover, the effect of intra-VTA leptin administration to reduce 4- and 20-h food intake and 20-h body weight was blocked by an inhibitor of Jak-2, at a dose that had no effect on food intake or body weight by itself, but not by local inhibition of either PI 3-kinase (LY-294002) or mTOR (rapamycin) in this timeframe. Taken together, these data support the hypothesis that leptin signaling in the VTA is involved in the regulation of energy balance, but, in contrast to the leptin signaling in the hypothalamus, these effects are mediated predominantly via Jak-2 signaling rather than via the IRS-PI 3-kinase or mTOR signaling pathway.

Impaired Energetic Metabolism After Central Leptin Signaling Leads to Massive Appendicular Bone Loss in Hindlimb-Suspended Rats

We previously showed in rats that the leptin effects on bone were dose dependent. Positive effects were observed when serum leptin concentration was in a physiological range. In contrast, important increases in serum leptin levels led to negative effects on bone formation similar to those reported after intracerebroventricular leptin administration in mice. To clarify whether leptin effects on bone depend on administration route and/or animal model, female rats were hindlimb unloaded or not and treated either with intracerebroventricular infusion of leptin or vehicle for 14 days. By increasing cerebrospinal fluid (CSF) leptin concentration, intracerebroventricular infusion of leptin significantly reduced food intake and consequently body weight, abdominal fat, and lean mass of the animals. Leptin infusion inhibited bone elongation over the 14 days and blunted cortical bone thickening at the femoral diaphysis site. Interestingly, leptin effects were site dependent in the cancellous bone envelopes, because tibia metaphysis BMD was lower and lumbar spine BMD was higher under intracerebroventricular leptin. Treated groups showed reduced bone remodeling independently of hindlimb unloading. Multiple downstream pathways were implicated in the mediation of these negative leptin effects on bone including not only stimulation of the sympathetic nervous system but also a decrease in somatotropic axis activity. Therefore, the intracerebroventricular leptin-induced bone loss could be largely related to the concurrent alteration of energetic and metabolic status. In summary, our study supports the hypothesis of a concentration-dependent balance between peripheral and central control of leptin on bone.

Impaired anorectic effect of leptin in neurotensin receptor 1-deficient mice

Neurotensin plays a role in regulating feeding behavior. Central injection of neurotensin reduces food intake and the anorectic effect of neurotensin is mediated through neurotensin receptor 1 (Ntsr1). Ntsr1-deficient mice are characterized by mild hyperphagia and overweight without hyperleptinemia. The mechanism by which Ntsr1-deficient mice develop these metabolic abnormalities is not well understood. Leptin, secreted by adipocytes, regulates food intake by acting on hypothalamic neurons including neurotensin-producing neurons. Since the anorectic effect of leptin is blocked by neurotensin receptor antagonist, we hypothesized that the anorectic effect of leptin is mediated through Ntsr1 in the central nervous system and that decreased sensitivity to the anorectic effect of leptin contributes to metabolic perturbations in Ntsr1-deficient mice. To address this hypothesis, we examined the effect of intracerebroventricular (i.c.v.) administration of leptin on food intake in Ntsr1-deficient mice. A single i.c.v. injection of leptin caused robust reductions in food intake in wild-type mice. These effects were markedly attenuated in Ntsr1-deficient mice. These data are consistent with our hypothesis that the anorectic effect of leptin is at least partly mediated through central Ntsr1 and that the leptin-Ntsr1 signaling pathway is involved in the regulation of food intake. Our data also suggest that the lack of Ntsr1 reduces sensitivity to the anorectic action of leptin, causing hyperphagia and abnormal weight gain.

Thyrotropin-Releasing Hormone Precursor Gene Knocking Down Impedes Melanocortin-Induced Hypertension in Rats

To the Editor: Recently, da Silva et al1 reported that endogenous melanocortin may cause elevation of arterial blood pressure (ABP) in spontaneously hypertensive rats. We invite authors to consider that the hypothalamic thyrotropin-releasing hormone (TRH) system may be involved, because spontaneously hypertensive rats show an hyperactivity of this system,2 and intracerebroventricular (ICV) injections of a prepro-TRH antisense oligonucleotide (AS) decreases both elevated TRH content and ABP independent of thyroid status.3 Leptin effects include increases in sympathetic activity …

Role of PI3 kinase in mediating renal sympathoactivation to leptin in obesity

Leptin acts in the central nervous system to decrease appetite and body weight and increase sympathetic nerve activity (SNA). Diet-induced obesity in mice is associated with resistance to the metabolic actions of leptin but preservation of its renal SNA and arterial pressure effects, leading to hypertension. The molecular basis of this selectivity in leptin resistance remains unknown. We have previously shown that leptin activation of renal SNA is phosphoinositol-3 kinase (PI3K)-dependent. Here, we tested the hypothesis that in obesity, renal sympathoactivation to leptin is mediated by PI3K. We compared the effect of intracerebroventricular (ICV) leptin on renal SNA in presence or absence of PI3K inhibitor, LY294002. Multifiber recording of SNA was performed under anesthesia (α-chloralose). Obesity was induced in C57BL/6J mice by exposure to a high fat diet (45%) for 20 weeks. After 20 weeks, mice on high-fat diet weighed ~20% more than those fed the normal diet. Weight of fat pads and plasma leptin were also significantly higher in the obese mice. The rise in renal SNA induced by ICV administration of leptin (5 μg) was of the same magnitude in both lean and obese mice. As expected, in lean mice ICV pre-treatment with LY294001 (0.1 μg) inhibited the increase in renal SNA induced by ICV leptin (Figure). Pre-treatment with LY294002 also blocked the increase in renal SNA induced by leptin in the obese mice (Figure). Our data confirm the pivotal role of PI3K in mediating the effects of leptin on renal SNA. Our findings also demonstrate the importance of PI3K as a key link between leptin receptor and renal SNA responses in obesity and provide a molecular substrate for selectivity in leptin resistance. Effect of ICV leptin (5 μg) on renal SNA in lean and obese mice in presence or absence of PI3K inhibitor, LY294002 (0.1 μg). ∗ P<0.05 vs. vehicle-leptin group, n=7–8 mice/group.

Hypothalamic Arcuate Nucleus Mediates the Sympathetic and Arterial Pressure Responses to Leptin

Leptin is an adipocyte-derived hormone that plays an important role in the regulation of energy homeostasis through its action in the central nervous system. Leptin decreases body weight by promoting satiety and increasing thermogenesis via increasing sympathetic nerve activity (SNA) to brown adipose tissue. Leptin also increases renal SNA and arterial pressure. The arcuate nucleus is considered as a major nucleus for leptin action on energy homeostasis. We tested whether leptin action in the arcuate nucleus simultaneously activates SNA to brown adipose tissue and the kidney. The sympathetic and cardiovascular responses to intra-arcuate injection of leptin were compared with those evoked by intracerebroventricular administration of leptin in rat. Intracerebroventricular administration of leptin (10 mug) caused a significant increase in SNA to brown adipose tissue and the kidney. Intracerebroventricular leptin also increased mean arterial pressure. Direct injection of leptin (500 ng) into the arcuate nucleus increased both brown adipose tissue (254+/-49%; P<0.001 versus vehicle) and renal SNA (111+/-31%; P<0.001 versus vehicle). Microinjection of leptin into the arcuate nucleus also produced a substantial increase in mean arterial pressure (from 82+/-3 to 100+/-7 mm Hg; P=0.02). These data demonstrate that leptin action in the arcuate nucleus of the hypothalamus is important for the control of sympathetic outflow to both brown adipose tissue and the kidney. These results also suggest that the cardiovascular effects of leptin might be evoked by the action of this hormone in the arcuate nucleus of the hypothalamus.

Perinatal overfeeding in rats results in increased levels of plasma leptin but unchanged cerebrospinal leptin in adulthood

To study the effect of perinatal programming and overfeeding on the hypothalamic control mechanisms of food intake in adult rats. Neonatal programming effects on body weight, food intake, central and peripheral leptin levels, hypothalamic neuropeptides, leptin receptors and central leptin responsiveness in adult rats. Plasma and cerebrospinal fluid (CSF) leptin levels were analyzed using radioimmunoassay. Neuropeptide mRNA levels were analyzed using in situ hybridization. Leptin receptor mRNA levels were analyzed using reverse transcriptase-polymerase chain reaction. Perinatally overfed rats growing up in small litters (SL) maintain their obese and hyperleptinemic phenotype in adulthood. However, leptin levels in CSF are abnormally low considering the plasmatic hyperleptinemia. In contrast to the already reported changes in perinatally overfed juvenile rats, perinatally overfed adult rats did not show any alteration in the expression of leptin receptor isoforms and evaluated neuropeptides. Moreover, SL adult rats showed a normal sensitivity regarding the inhibitory effect of intracerebroventricular leptin administration on food intake. Perinatal overfeeding does not induce alterations in either the anorectic response to central leptin administration or expression of leptin receptors and neuropeptides in adulthood. The leptin resistance to peripheral leptin in SL adult rats may be related to impaired leptin transport across the blood-brain barrier.

The Role of Intracerebroventricular Administration of Leptin in the Stimulation of Prothyrotropin Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus

We previously have shown that leptin regulates proTRH in the paraventricular nucleus (PVN) of the hypothalamus through two pathways. The first one acts directly on proTRH neurons, and the second one (indirect) acts through the melanocortin system (arcuate nucleus). However, it is unknown whether the direct or the indirect pathways of leptin action on proTRH neurons occurs on separated or on the same subsets of neurons within the PVN region. We used immunostaining for the phosphorylated signal transducer and activator of transcription 3 to localize direct leptin signaling, and the phosphorylated cAMP response element binding protein to localize indirect signaling on proTRH neurons in animals intracerebroventricularly injected with leptin. With this approach we were able to identify two subsets of neuronal populations responsive to leptin, which are distributed in different regions within the PVN. ProTRH neurons directly responsive to leptin were located mainly in the medial and posterior part of the PVN, and they were not primarily related to the hypothalamic pituitary thyroid axis. Whereas, proTRH neurons indirectly responsive (through alpha-MSH) to leptin were located mainly in the anterior, medial, and periventricular part of the PVN, and related to the hypothalamic pituitary thyroid axis. In addition, alpha-MSH showed to affect the processing of proTRH and up-regulated the prohormone convertase 1/3. In this study, we show evidence supporting the hypothesis that in the PVN there are subpopulations of proTRH neurons responding to leptin, which is dependent upon the way leptin reaches its primary target(s) in the hypothalamus. These findings are critical to a better understanding of leptin-mediated actions in energy expenditure.