Systemic Leptin Administration Research Articles

Ablation of Nampt in AgRP neurons leads to neurodegeneration and impairs fasting- and ghrelin-mediated food intake.

Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulates food intake and whole-body metabolism. NAD+ regulates multiple cellular processes controlling energy metabolism. Yet, its role in hypothalamic AgRP neurons to control food intake is poorly understood. Here, we aimed to assess whether genetic deletion of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+ production, affects AgRP neuronal function to impact whole-body metabolism and food intake. Metabolic parameters during fed and fasted states, and upon systemic ghrelin and leptin administration were studied in AgRP-specific Nampt knockout (ARNKO) mice. We monitored neuropeptide expression levels and density of AgRP neurons in ARNKO mice from embryonic to adult age. NPY cells were used to determine effects of NAMPT inhibition on neuronal viability, energy status, and oxidative stress in vitro. In these cells, NAD+ depletion reduced ATP levels, increased oxidative stress, and promoted cell death. Agrp expression in the hypothalamus of ARNKO mice gradually decreased after weaning due to progressive AgRP neuron degeneration. Adult ARNKO mice had normal glucose and insulin tolerance, but exhibited an elevated respiratory exchange ratio (RER) when fasted. Remarkably, fasting-induced food intake was unaffected in ARNKO mice when evaluated in metabolic cages, but fasting- and ghrelin-induced feeding and body weight gain decreased in ARNKO mice when evaluated outside metabolic cages. Collectively, deletion of Nampt in AgRP neurons causes progressive neurodegeneration and impairs fasting and ghrelin responses in a context-dependent manner. Our data highlight an essential role of Nampt in AgRP neuron function and viability.

A Leptin-Mediated Neural Mechanism Linking Breathing to Metabolism

Breathing is coupled to metabolism. Leptin, a peptide mainly secreted in proportion to adipose tissue mass, increases energy expenditure with a parallel increase in breathing. We demonstrate that optogenetic activation of LepRb neurons in the nucleus of the solitary tract (NTS) mimics the respiratory stimulation after systemic leptin administration. We show that leptin activates the sodium leak channel (NALCN), thereby depolarizing a subset of glutamatergic (VGluT2) LepRb NTS neurons expressing galanin. Mice with selective deletion of NALCN in LepRb neurons have increased breathing irregularity and central apneas. On a high-fat diet, these mice gain weight with an associated depression of minute ventilation and tidal volume, which are not detected in control littermates. Anatomical mapping reveals LepRb NTS-originating glutamatergic axon terminals in a brainstem inspiratory premotor region (rVRG) and dorsomedial hypothalamus. These findings directly link a defined subset of NTS LepRb cells to the matching of ventilation to energy balance.

Perineuronal Net Formation during the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus.

SummaryIn leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homeostasis2,3. As this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period (CP) for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, CP closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans as well as rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed by PNN-like structures. In mice, these neurons are densely-packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both GABAergic and leptin receptor-positive, including a majority of Agrp neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the CP for Agrp neuron maturation and is dependent on input from circulating leptin, as postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the CP. We conclude that neurons crucial to metabolic homeostasis are enmeshed by PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC-ME junction, where metabolically-relevant humoral signals are sensed.

Effects of Leptin on the Skeleton.

Leptin originates in adipocytes, including those in bone marrow, and circulates in concentrations 20 to 90 times higher than those in the cerebrospinal fluid. It has direct anabolic effects on osteoblasts and chondrocytes, but it also influences bone indirectly, via the hypothalamus and sympathetic nervous system, via changes in body weight, and via effects on the production of other hormones (e.g., pituitary). Leptin's role in bone physiology is determined by the balance of these conflicting effects. Reflecting this inconsistency, the leptin-deficient mouse has reduced length and bone mineral content of long bones but increased vertebral trabecular bone. A consistent bone phenotype in human leptin deficiency has not been established. Systemic leptin administration in animals and humans usually exerts a positive effect on bone mass, and leptin administration into the cerebral ventricles usually normalizes the bone phenotype in leptin-deficient mice. Reflecting the role of the sympathetic nervous system in mediating the central catabolic effects of leptin on the skeleton, β-adrenergic agonists and antagonists have major effects on bone in mice, but this is not consistently seen in humans. The balance of the central and peripheral effects of leptin on bone remains an area of substantial controversy and might vary between species and according to other factors such as body weight, baseline circulating leptin levels, and the presence of specific pathologies. In humans, leptin is likely to contribute to the positive relationship observed between adiposity and bone density, which allows the skeleton to respond appropriately to changes in soft tissue mass.

Systemic leptin administration alters callus VEGF levels and enhances bone fracture healing in wildtype and ob/ob mice

IntroductionLeptinʼs role in bone formation has been reported, however, its mechanism of affecting bone metabolism is remaining unclear. In this study, we aimed to test whether leptin has a positive effect on fracture healing through the possible mechanism of increasing vascular endothelial growth factor (VEGF) expression in callus tissue. MethodsStandardized femur fractures were created in leptin-deficient ob/ob and wildtype C57BL/6J mice, and recombinant mouse leptin or its vehicle (physiological saline) was administered intraperitoneally during the study. Body weight, radiological, histologic and immunoblotting analyses were performed at different stages of fracture healing. Key findingsThe results showed that leptin treatment led to lower rate of body weight change in both mice genotypes. Radiological and histological analyses showed that the experimental groups had better fracture healing at 14, 21 and 28 days compared to the control groups. Leptin-treated groups had significantly higher VEGF expression in callus compared with the control groups at 2 and 3 weeks post-fracture except normal mice at 2 weeks, and leptin-deficient mice had higher VEGF levels in calluses than normal mice at the same timepoint. ConclusionLow-dose systemically-administered leptin has a positive effect on promoting fracture healing during the latter stages in a clinically-relevant mouse bone fracture model, and increase callus VEGF levels.

Phenotypic effects of an induced mutation of the ObRa isoform of the leptin receptor

Leptin receptors play critical roles in mediating leptin's pleiotropic effects on mammalian physiology. To date, six splice variants of the leptin receptor gene have been identified [1-3]. These splice variants have identical extracellular leptin binding motifs but different intracellular C termini. The finding that mutations specifically ablating the function of ObRb cause obesity has established a critical role for this isoform in leptin signaling [1,7]. ObRa is the most abundant splicing isoform with a broad tissue distribution [5], and it has been proposed to play roles in regulating leptin bioavailability, CSF (cerebrospinal fluid) transport and function by forming heterodimers with ObRb and also activating signal transduction via JAK2 in-vitro [5-10]. To assess the in-vivo role of ObRa, we generated an ObRa KO mouse by deleting the ObRa-specific exon 19a. Homozygous mutant mice breed normally and are indistinguishable from wild-type mice on regular chow diet, but show a slightly increased basal plasma leptin, a slight improvement of their GTT and a slightly reduced response to systemic leptin administration. These mice also show a modest but statistically significant increase in weight when placed on a high fat diet with a slightly reduced CSF/plasma ratio of leptin. These data suggest that ObRa plays a role in mediating some of leptin's effects but that the phenotypic consequences are modest compared to a deletion of ObRb.

Systemic leptin produces a long-lasting increase in respiratory motor output in rats

Leptin decreases food intake and increases energy expenditure. Leptin administration into the CNS of mice or rats increases alveolar ventilation and dysfunction in leptin signaling has been implicated in the hypoventilation that can accompany obesity. An increase in CO2 chemosensitivity has been implicated in this response but it is unclear whether ventilation is augmented when PCO2 is maintained constant. We examined the effects of intravenous leptin to test the hypothesis that systemic leptin administration in isoflurane anesthetized, mechanically ventilated and vagotomized rats would lead to a sustained increase in respiratory motor output that was independent of changes in end-tidal PCO2, body temperature or lung inflation pressure (an indicator of overall lung and chest wall compliance). In anesthetized Sprague-Dawley rats with end-tidal PCO2, lung compliance and rectal temperature maintained constant, injection of a bolus of leptin (0.25 mg, 0.5 mg/ml, i.v.), followed over the next 1 h by the intravenous infusion of an additional 0.25 mg, elicited a progressive increase in the peak amplitude of integrated phrenic nerve discharge lasting at least 1 h beyond the end of the infusion. The increase peaked at 90 min at 58.3 ± 5.7% above baseline. There was an associated increase in the slope of the phrenic response to increasing inspired CO2. There was also a moderate and sustained decrease in arterial pressure 9 ± 1.3 mmHg at 120 min, with no associated change in heart rate. These data indicate that leptin elicits a sustained increase in respiratory motor output that outlasts the administration leptin via a mechanism that does not require alterations in arterial PCO2, body temperature, or systemic afferent feedback via the vagus nerves. This stimulation may help to prevent obesity-related hypoventilation.

Systemic Leptin Administration in Supraphysiological Doses Maintains Bone Mineral Density and Mechanical Strength Despite Significant Weight Loss

The effects of leptin on bone are controversial. Although in vitro studies have shown that leptin stimulates osteoblast differentiation and mineralization and inhibits osteoclastogenesis, some rodent studies have shown that leptin administered centrally might result in decreased bone formation. In the present study we have investigated the skeletal effects of supraphysiological concentrations of leptin administered sc to rats. Female Fischer rats were given leptin 100 μg/d, 200 μg/d, or saline by continuous infusion for 9 wk. Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry, bone microarchitecture was analyzed by micro-computed tomography, and biomechanical properties were tested by three-point bending experiments. At the end of the study, the body weight was significantly lower in rats receiving leptin compared with controls (-10.8% and -12.0% in low- and high-dose leptin groups, respectively). The high-dose leptin group also significantly lost weight compared with baseline. The plasma leptin concentration was 14- and 33-fold increased in the low- and high-dose groups, respectively. No significant differences in femoral BMD were observed. Whole-body BMD was significantly lower in the low-dose leptin group, whereas there was no difference between the high-dose leptin group and the control. Mechanical strength and microarchitecture were similar in the high-dose and the control group. The low-dose group, however, had decreased cortical volume in the femoral metaphysis, lowered bone strength, and altered moment of inertia. In conclusion, leptin given at very high doses maintains BMD, microarchitecture, and mechanical strength in female rats, despite a significant decrease in body weight.

Cardiovascular Actions of Leptin in the Subfornical Organ are Abolished by Diet‐Induced Obesity

The subfornical organ (SFO), a sensory circumventricular organ lacking the normal blood-brain barrier with well documented roles in cardiovascular regulation, has recently been identified as a potential site at which the adipokine, leptin, may act to influence central autonomic pathways. Systemic and central leptin administration has been shown to increase blood pressure and it has been suggested that selective leptin resistance contributes to obesity-related hypertension. Given the relationship between obesity and hypertension, the present study aimed to investigate the cardiovascular consequences of the direct administration of leptin into the SFO of young lean rats and in the diet-induced obesity (DIO) rat model, which has been shown to be leptin-resistant. Leptin administration (500 fmol) directly into the SFO of young rats resulted in rapid decreases in blood pressure (BP) [mean area under the curve (AUC) = -677.8 ± 167.1 mmHg*s; n = 9], without an effect on heart rate (mean AUC = -21.2 ± 13.4 beats; n = 9), and these effects were found to be dose-related as microinjection of 5 pmol of leptin into the SFO had a larger effect on BP (mean AUC = -972.3 ± 280.1 mmHg*s; n = 4). These BP effects were also shown to be site-specific as microinjection of leptin into non-SFO regions or into the ventricle was without effect on BP (non-SFO: mean AUC = -22.4 ± 55.3 mmHg*s; n = 4; ventricle: mean AUC = 194.0 ± 173.0 mmHg*s; n = 6). By contrast, microinjection of leptin into leptin-resistant DIO rats was without effect on BP (mean AUC = 205.2 ± 75.1 mmHg*s; n = 4). These observations suggest that the SFO may be an important relay centre through which leptin, in normal weight, leptin responsive rats, acts to maintain BP within normal physiological limits through descending autonomic pathways involved in cardiovascular control and that, in obese, leptin-resistant, rats leptin no longer influences SFO neurones, resulting in an elevated BP, thus contributing to obesity-related hypertension.

Intermittent hypoxia and systemic leptin administration induces pSTAT3 and Fos/Fra-1 in the carotid body

Glomus cells within the carotid body are known to respond to hypoxic stimuli. Recently, these cells have been shown to express the long form of the leptin receptor (Ob-Rb). However, whether these glomus cells expressing the Ob-Rb are activated by hypoxic stimuli is not known. Therefore, in this study we investigated whether intermittent hypoxia (IH) or changes in circulating levels of leptin induced phosphorylated signal transducer and activator of transcription 3 (pSTAT3), the immediate early gene c-fos protein, or fos-related antigen-1 protein (Fra-1) within carotid body glomus cells that expressed the Ob-Rb, and within neurons of the petrosal (PG) and nodose (NG) ganglia. Rats were subjected to IH (120s normoxia, 80s hypoxia for 8h) or normoxia (8h), or intravenous injections of leptin (50 or 200ng/0.1mL) or the vehicle saline. Plasma leptin levels were measured in animals exposed to IH and normoxia. Exposure to 8h of IH increased plasma leptin levels greater than 2-fold compared to normoxic controls. Animals were then perfused with Zamboni's fixative, and the region of the carotid bifurcation containing the carotid body and PG/NG complex was removed, paraffin embedded and sectioned at 6μm for immunohistochemical processing. Carotid body glomus cells were identified by their expression of tyrosine hydroxylase immunoreactivity. These glomus cells also expressed the OB-Rb and were found to express pSTAT3-, fos-, and Fra-1-like immunoreactivity in response to both IH and systemic leptin injections. IH and leptin injections also increased fos and Fra-1 like expression in the PG, NG and jugular ganglion. Taken together, these data suggest IH alters circulating leptin which in turn activates directly carotid body glomus cells to exert a modulatory effect on the peripheral chemoreceptor reflex.

Leptin Signaling Modulates the Activity of Urocortin 1 Neurons in the Mouse Nonpreganglionic Edinger-Westphal Nucleus

A recent study systematically characterized the distribution of the long form of the leptin receptor (LepRb) in the mouse brain and showed substantial LepRb mRNA expression in the nonpreganglionic Edinger-Westphal nucleus (npEW) in the rostroventral part of the midbrain. This nucleus hosts the majority of urocortin 1 (Ucn1) neurons in the rodent brain, and because Ucn1 is a potent satiety hormone and electrical lesioning of the npEW strongly decreases food intake, we have hypothesized a role of npEW-Ucn1 neurons in leptin-controlled food intake. Here, we show by immunohistochemistry that npEW-Ucn1 neurons in the mouse contain LepRb and respond to leptin administration with induction of the Janus kinase 2-signal transducer and activator of transcription 3 pathway, both in vivo and in vitro. Furthermore, systemic leptin administration increases the Ucn1 content of the npEW significantly, whereas in mice that lack LepRb (db/db mice), the npEW contains considerably reduced amount of Ucn1. Finally, we reveal by patch clamping of midbrain Ucn1 neurons that leptin administration reduces the electrical firing activity of the Ucn1 neurons. In conclusion, we provide ample evidence for leptin actions that go beyond leptin's well-known targets in the hypothalamus and propose that leptin can directly influence the activity of the midbrain Ucn1 neurons.

Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling

The first intron of FTO contains common single nucleotide polymorphisms associated with body weight and adiposity in humans. In an effort to identify the molecular basis for this association, we discovered that FTO and RPGRIP1L (a ciliary gene located in close proximity to the transcriptional start site of FTO) are regulated by isoforms P200 and P110 of the transcription factor, CUX1. This regulation occurs via a single AATAAATA regulatory site (conserved in the mouse) within the FTO intronic region associated with adiposity in humans. Single nucleotide polymorphism rs8050136 (located in this regulatory site) affects binding affinities of P200 and P110. Promoter-probe analysis revealed that binding of P200 to this site represses FTO, whereas binding of P110 increases transcriptional activity from the FTO as well as RPGRIP1L minimal promoters. Reduced expression of Fto or Rpgrip1l affects leptin receptor isoform b trafficking and leptin signaling in N41 mouse hypothalamic or N2a neuroblastoma cells in vitro. Leptin receptor clusters in the vicinity of the cilium of arcuate hypothalamic neurons in C57BL/6J mice treated with leptin, but not in fasted mice, suggesting a potentially important role of the cilium in leptin signaling that is, in part, regulated by FTO and RPGRIP1L. Decreased Fto/Rpgrip1l expression in the arcuate hypothalamus coincides with decreased nuclear enzymatic activity of a protease (cathepsin L) that has been shown to cleave full-length CUX1 (P200) to P110. P200 disrupts (whereas P110 promotes) leptin receptor isoform b clustering in the vicinity of the cilium in vitro. Clustering of the receptor coincides with increased leptin signaling as reflected in protein levels of phosphorylated Stat3 (p-Stat3). Association of the FTO locus with adiposity in humans may reflect functional consequences of A/C alleles at rs8050136. The obesity-risk (A) allele shows reduced affinity for the FTO and RPGRIP1L transcriptional activator P110, leading to the following: 1) decreased FTO and RPGRIP1L mRNA levels; 2) reduced LEPR trafficking to the cilium; and, as a consequence, 3) a diminished cellular response to leptin.

Leptin Augments Cerebral Hemodynamic Reserve after Three-Vessel Occlusion: Distinct Effects on Cerebrovascular Tone and Proliferation in a Nonlethal Model of Hypoperfused Rat Brain

The adipocytokine leptin has distinct functions regulating vascular tone, inflammation, and collateral artery growth. Arteriogenesis is an inflammatory process and provides a mechanism to overcome the effects of vascular obstruction. We, therefore, tested the effects of leptin in hypoperfused rat brain (three-vessel occlusion). Systemic leptin administration for 1 week after occlusion surgery increased cerebral hemodynamic reserve similar to granulocyte-macrophage colony-stimulating factor (GM-CSF), as indicated by improved CO(2) reactivity (vehicle 0.53%±0.26% versus leptin 1.05%±0.6% per mm Hg arterial pCO(2), P<0.05). Infusion of microspheres under maximal vasodilation failed to show a positive effect of leptin on cerebral perfusion (vehicle 64.9%±4.5% versus leptin 66.3%±7.0%, occluded/nonoccluded hemisphere). Acute treatment with GM-CSF led to a significant increased CO(2) reactivity and cerebral perfusion (79.2%±8.1% versus 64.9%±4.5%, P<0.05). Vasoconstrictive response of isolated rat carotid artery rings, after phenylephrine was attenuated at 24 hours following preincubation with leptin, was unaffected by removal of endothelium but abrogated by coculture with N-(omega)-nitro-L-arginine methylester, pointing toward an inducible nitric oxide synthase-mediated mechanism. In chronic cerebral hypoperfusion, acute leptin treatment restored the hemodynamic reserve of the cerebral vasculature through its effects on vascular tone, while leaving vascular outward remodeling unaffected. Our results, for the first time, reveal a protective role of leptin on vascular function in hemodynamically compromised brain tissue.

Nuclear translocation of the transcription factor STAT5 in the rat brain after systemic leptin administration

Leptin binding to its functional receptor stimulates JAK-STAT-signaling pathway, which finally results in activation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (STAT) family, namely of STAT3. Here we report for the first time that systemic treatment with leptin (5 mg/kg; intraperitoneal injection) also increased the number of nuclear STAT5 signals in the hypothalamus. In particular, the entire arcuate nucleus (ARC), the ventral premammilary nucleus (PMV), and the supraoptic nucleus (SO) showed an enhanced nuclear STAT5 translocation in response to leptin when compared to saline, 120 min after the respective injection. Co-localization studies revealed that a high percentage of those STAT5-responsive cells proved to be neurons. In addition, some astrocytes within the ARC showed nuclear STAT5 signals. The functional relevance of leptin-induced nuclear STAT5 activation in hypothalamic cells still has to be determined.

Severely Impaired Insulin Signaling in Chronic Wounds of Diabetic ob/ob Mice : A Potential Role of Tumor Necrosis Factor-α

Wound-healing disorders are major complications of diabetes mellitus. Here, we investigated insulin-mediated signaling in nonwounded skin and in cutaneous tissue regeneration of healthy C57BL/6 and diabetes-impaired leptin-deficient obese/obese (ob/ob) mice. The insulin receptor (InsR) was abundantly expressed in wound margins and granulation tissue during acute healing in healthy mice. Remarkably, active signaling from the InsR, as assessed by phosphorylation of downstream targets such as protein tyrosine phosphatase-1B, glycogen synthase (GS), and GS kinase, was nearly absent in nonwounded and acutely healing skin from ob/ob mice. Systemic leptin administration to ob/ob mice reverted the diabetic phenotype and improved tissue regeneration as well as the impaired expression of InsR, insulin receptor substrate-1 and insulin receptor substrate-2, and downstream signaling (phosphorylation of GS kinase and GS) in late wounds and nonwounded skin of ob/ob mice. Importantly, tumor necrosis factor (TNF)-alpha was a mediator of insulin resistance in keratinocytes in vitro and in ob/ob wound tissue in vivo. Systemic administration of a monoclonal anti-TNF-alpha antibody (V1q) in wounded ob/ob mice attenuated wound inflammation, improved re-epithelialization, and restored InsR expression and signaling in wound tissue of ob/ob mice. These data suggest that InsR signaling in diabetes-impaired wounds is sensitive to inflammatory conditions and that anti-inflammatory approaches, such as anti-TNF-alpha strategies, improve diabetic wound healing.

The effect of systemic leptin administration on aorta smooth muscle responses in diabetic rats

Leptin produces effects in central nervous system and peripheral tissues via its specific receptors. Leptin also stimulates nitric oxide release in a concentration-dependent manner. In this study, our aim was to test the hypothesis that whether leptin has a modulatory role on endothelium or smooth muscle function in streptozotocin (STZ)-induced diabetic rats. Wistar-Albino rats were divided into four groups: 1 -- Control, 2 -- Diabetic, 3 -- Control + leptin and 4 -- Diabetic + leptin. Experimental diabetes was produced by intraperitoneal injection of a single dose of STZ (55 mg/kg). Diabetes was determined by increased fasting blood glucose level on the 7th day of the experiment. Leptin (0.1 mg/kg/day) was administered intraperitoneally for 5 days. At the end of the 5th day, thoracic aortas were isolated and phenylephrine (Phe)-induced contractions and acetylcholine (ACh)-induced relaxations of each group were estimated. In diabetic rats, Phe-induced contractility was increased (p < 0.05). Leptin pre-treatment increased the Phe-induced contractility significantly in aortic rings obtained from diabetic rats (p < 0.05). In normal rats, leptin administration produced only a slight and non-significant increase in Phe-induced contractions. Although the relaxant responses were decreased in diabetic rats, leptin administration enhanced the ACh-induced relaxation in both normal and diabetic animals significantly. As a conclusion; chronic leptin pre-treatment caused a significant increase both in Phe-induced contractions and ACh-induced Endothelial-Derived Relaxing Factor (EDRF)/Nitric oxide-mediated relaxations in the aortic rings isolated from streptozotocin-induced diabetic rats. This peptide hormone caused a significant increase in the relaxations obtained by ACh while not inducing a significant alteration in the contractile effect of Phe in control rats.

Physiological regulation of hypothalamic IL-1beta gene expression by leptin and glucocorticoids: implications for energy homeostasis.

Interleukin-1beta (IL-1beta) is synthesized in a variety of tissues, including the hypothalamus, where it is implicated in the control of food intake. The current studies were undertaken to investigate whether hypothalamic IL-1beta gene expression is subject to physiological regulation by leptin and glucocorticoids (GCs), key hormones involved in energy homeostasis. Adrenalectomy (ADX) increased hypothalamic IL-1beta mRNA levels twofold, measured by real-time PCR (P < 0.05 vs. sham-operated controls), and this effect was blocked by subcutaneous infusion of a physiological dose of corticosterone. Conversely, hypothalamic IL-1beta mRNA levels were reduced by 30% in fa/fa (Zucker) rats, a model of genetic obesity caused by leptin receptor mutation (P = 0.01 vs. lean littermates), and the effect of ADX to increase hypothalamic IL-1beta mRNA levels in fa/fa rats (P = 0.02) is similar to that seen in normal animals. Moreover, fasting for 48 h (which lowers leptin and raises corticosterone levels) reduced hypothalamic IL-1beta mRNA levels by 30% (P = 0.02), and this decrease was fully reversed by refeeding for 12 h. Thus leptin and GCs exert opposing effects on hypothalamic IL-1beta gene expression, and corticosterone plays a physiological role to limit expression of this cytokine in both the presence and absence of intact leptin signaling. Consistent with this hypothesis, systemic leptin administration to normal rats (2 mg/kg ip) increased hypothalamic IL-1beta mRNA levels twofold (P < 0.05 vs. vehicle), an effect similar to that of ADX. These data support a model in which expression of hypothalamic IL-1beta is subject to opposing physiological regulation by corticosterone and leptin.

Modulatory role of testosterone in plasma leptin turnover in rats.

We explored whether testosterone influences circulating leptin turnover in rats. Sham-operated male and female rats and 21-d gonadectomized rats treated or not treated with testosterone propionate were used. Anesthetized rats were implanted with an iv catheter, and then blood samples were drawn before and throughout a 60-min period following systemic leptin administration. Plasma testosterone, estradiol, and leptin concentrations were monitored. The results indicated that while gonadectomy blunted circulating concentrations of the homologous sex steroid, testosterone therapy, in gonadectomized rats, restored plasma testosterone concentrations to values found in normal male rats. Pharmacokinetic parameters evaluated during the test indicated the following: First, in the overall pharmacokinetic analyses, testosterone therapy in gonadectomized rats induced a more rapid disappearance of leptin from the circulation. Second, orchidectomy significantly enhanced the area under the curve (AUC) of circulating leptin, an effect fully reversed by testosterone treatment. Third, testosterone treatment in ovariectomized rats significantly decreased the AUC of leptin concentrations. Fourth, while gonadectomy alone did not modify circulating leptin half-life, conversely, testosterone therapy in gonadectomized rats decreased leptin halflife in the circulation. Finally, while orchidectomy reduced leptin body clearance, this parameter was increased by androgen therapy in gonadectomized rats. Our results strongly support that testosterone could play a main role in plasma leptin turnover by increasing leptin clearance rate and shortening plasma leptin half-life.

Leptin: A new growth factor for the small intestine

Purpose: This study was designed to evaluate the potential growth factor effects of systemic administration of leptin on mucosal mass and absorptive function in normal rat intestine. Methods: Twenty male Sprague-Dawley rats underwent placement of a jugular venous catheter connected to a subcutaneous osmotic pump designed to deliver its contents at a constant rate. The rats were divided into 4 groups (n = 5 per group) based on the contents of the osmotic pump: group 1, 0.1% bovine serum albumin; group 2, leptin, 6.25 [mu ]g/kg/d; Group 3, leptin, 18.75 [mu ]g/kg/d; Group 4, leptin, 43.75 [mu ]g/kg/d. After a 14-day infusion, [ 14C] galactose and [ 14C] glycine absorption were determined using a closed, recirculation technique. DNA content was determined from mucosal biopsies. Total RNA was extracted from mucosal samples, reverse transcribed, and amplified via polymerase chain reaction for the following primer pairs: sodium/glucose cotransporter (SGLT-1), fructose transporter (GLUT-5), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal standard). Statistical analysis was performed by analysis of variance and expressed as mean [plusmn] SEM. Results: Systemic administration of increasing doses of leptin enhanced DNA content when compared with the appropriate control (group 2, 1.06 [plusmn] 0.04 [ P [lt ] .05]; group 3, 1.1 [plusmn] 0.05 [ P [lt ] .01]; and group 4, 1.07 [plusmn] 0.06 [ P [lt ] .05]. Leptin enhanced mucosal absorptive function (galactose: group 2, 2.31 [plusmn] 0.15 [ P [lt ] .01]; group 3, 2.71 [plusmn] 0.06 [ P [lt ] .01]; group 4, 2.19 [plusmn] 0.28 [ P [lt ] .05]; glycine: group 2, 2.34 [plusmn] 0.31 [ P [lt ] .05]; group 3, 3.32 [plusmn] 0.14 [ P [lt ] .01]; group 4, 3.1 [plusmn] 0.27 [ P [lt ] .01]) in the normal intestine when compared with the appropriate control animals. Also, leptin enhanced the gene expression of the carbohydrate transporters when compared with the appropriate control rats. Conclusions: These data show that systemic leptin administration enhances mucosal mass and absorptive function in normal rat intestine. Thus, leptin appears to be a growth factor for normal small intestine and may play a role in patients who acquire intestinal dysfunction.

Novel Effect of Leptin on Small Intestine Adaptation

Leptin is a 16-kDa peptide produced by adipocytes that plays an important role in the regulation of body fat and satiety. We have previously shown that leptin is a growth factor for normal rat small intestine. This study was designed to examine the effect of systemic leptin administration on small bowel absorptive function after massive small bowel resection (MSBR). Twenty-one adult male Sprague-Dawley rats underwent an 80% small bowel resection and end-to-end jejunoileal anastomosis. Seven days following resection, all rats had placement of a jugular venous catheter connected to a subcutaneously placed osmotic minipump and were divided into three groups based on the content of each minipump: Group 1 (n = 7) 0.1% bovine serum albumin; Group 2 (n = 7) leptin 2 microg/kg/day; and Group 3 (n = 7) leptin 4 microg/kg/day. Following a 14-day infusion period, [(14)C]galactose absorption was measured using a closed-recirculation technique. Mucosal DNA content was determined for all groups using a standard DNA purification kit. Mucosal RNA was extracted and RT-PCR was performed using the following primers: sodium/glucose cotransporter (SGLT-1), fructose transporter (GLUT-5), and glyceraldehyde-3-phosphate dehydrogenase, an internal standard. PCR products were separated on a 4% agarose gel and relative band intensities were measured. Statistical analysis was performed using ANOVA and expressed as means +/- SEM. Group 2 showed a 44% increase in galactose absorption (P < 0.01) and a 14% increase in GLUT-5 band intensity (P < 0.05), but no change in DNA content or SGLT band intensity. This study demonstrates for the first time that leptin enhances small intestine carbohydrate absorption beyond the normal adaptive response following MSBR. Leptin may be clinically useful in patients with inadequate intestinal function.