Leptin Infusion Research Articles

Deletion of protein tyrosine phosphatase 1b in proopiomelanocortin neurons reduces neurogenic control of blood pressure and protects mice from leptin- and sympatho-mediated hypertension

Protein tyrosine phosphatase 1b (Ptp1b), which represses leptin signaling, is a promising therapeutic target for obesity. Genome wide deletion of Ptp1b, increases leptin sensitivity, protects mice from obesity and diabetes, but alters cardiovascular function by increasing blood pressure (BP). Leptin-control of metabolism is centrally mediated and involves proopiomelanocortin (POMC) neurons. Whether these neurons contribute to leptin-mediated increases in BP remain unclear. We hypothesized that increasing leptin signaling in POMC neurons with Ptp1b deletion will sensitize the cardiovascular system to leptin and enhance neurogenic control of BP. We analyzed the cardiovascular phenotype of Ptp1b+/+ and POMC-Ptp1b−/− mice, at baseline and after 7 days of leptin infusion or sympatho-activation with phenylephrine. POMCPtp1b deletion did not alter baseline cardiovascular hemodynamics (BP, heart rate) but reduced BP response to ganglionic blockade and plasma catecholamine levels that suggests a decreased neurogenic control of BP. In contrast, POMC-Ptp1b deletion increased vascular adrenergic reactivity and aortic α-adrenergic receptors expression. Chronic leptin treatment reduced vascular adrenergic reactivity and blunted diastolic and mean BP increases in POMC-Ptp1b−/− mice only. Similarly POMC-Ptp1b−/− mice exhibited a blunted increased in diastolic and mean BP accompanied by a gradual reduction in adrenergic reactivity in response to chronic vascular sympatho-activation with phenylephrine. Together these data rule out our hypothesis but suggest that deletion of Ptp1b in POMC neurons protects from leptin- and sympatho-mediated increases in BP. Vascular adrenergic desensitization appears as a protective mechanism against hypertension, and POMC-Ptp1b as a key therapeutic target for the treatment of metabolic and cardiovascular dysfunctions associated with obesity.

Leptin acts in the brain to influence hypoglycemic counterregulation: disparate effects of acute and recurrent hypoglycemia on glucagon release.

Leptin has been shown to diminish hyperglycemia via reduced glucagon secretion, although it can also enhance sympathoadrenal responses. However, whether leptin can also inhibit glucagon secretion during insulin-induced hypoglycemia or increase epinephrine during acute or recurrent hypoglycemia has not been examined. To test whether leptin acts in the brain to influence counterregulation, hyperinsulinemic hypoglycemic (∼45 mg/dl) clamps were performed on rats exposed to or not exposed to recurrent hypoglycemia (3 days, ∼40 mg/dl). Intracerebroventricular artificial cerebral spinal fluid or leptin was infused during the clamp. During acute hypoglycemia, leptin decreased glucagon responses by 51% but increased epinephrine and norepinephrine by 24 and 48%, respectively. After recurrent hypoglycemia, basal plasma leptin levels were undetectable. Subsequent brain leptin infusion during hypoglycemia paradoxically increased glucagon by 45% as well as epinephrine by 19%. In conclusion, leptin acts within the brain to diminish glucagon secretion during acute hypoglycemia but increases epinephrine, potentially limiting its detrimental effects during hypoglycemia. Exposure to recurrent hypoglycemia markedly suppresses plasma leptin, whereas exogenous brain leptin delivery enhances both glucagon and epinephrine release to subsequent hypoglycemia. These data suggest that recurrent hypoglycemia may diminish counterregulatory responses in part by reducing brain leptin action.

Leptin/LepRb in the Ventral Tegmental Area Mediates Anxiety-Related Behaviors.

Background:Leptin, an adipose-derived hormone, has been implicated in emotional regulation. We have previously shown that systemic administration of leptin produces anxiolytic-like effects and deletion of the leptin receptor, LepRb, in midbrain dopamine neurons leads to an anxiogenic phenotype. This study investigated whether activation or deletion of LepRb in the ventral tegmental area of adult mice is capable of inducing anxiolytic and anxiogenic effects, respectively.Methods:Mice were cannulated in the ventral tegmental area and received bilateral intra-ventral tegmental area infusions of leptin or the JAK2/STAT3 inhibitor AG490. Anxiety-like behaviors were assessed using the elevated plus-maze, light-dark box, and novelty suppressed feeding tests. Deletion of LepRb in the ventral tegmental area was achieved by bilateral injection of AAV-Cre into the ventral tegmental area of adult Leprflox/flox mice. Anxiety-related behaviors were evaluated 3 weeks after viral injection.Results:Intra-ventral tegmental area infusions of leptin reduced anxiety-like behaviors, as indicated by increased percent open-arm time and open-arm entries in the elevated plus-maze test, increased time spent in the light side and decreased latency to enter the light side of the light-dark box, and decreased latency to feed in the novelty suppressed feeding test. Blockade of JAK2/STAT3 signaling in the ventral tegmental area by AG490 attenuated the anxiolytic effect produced by systemic administration of leptin. Leprflox/flox mice injected with AAV-Cre into the ventral tegmental area showed decreased leptin-induced STAT3 phosphorylation and enhanced anxiety-like behaviors in the elevated plus-maze test and the novelty suppressed feeding test.Conclusions:These findings suggest that leptin-LepRb signaling in the ventral tegmental area plays an important role in the regulation of anxiety-related behaviors.

Abstract P084: Oxidative Stress Preconditioning Is Required for Leptin to Stimulate Renin Release From Mouse Juxtaglomerular Cells

Leptin is enhanced in animal models of obesity. The role of leptin in hypertension is unclear with some studies showing antihypertensive actions and others showing a pro-hypertensive role. In normotensive, non-obese, animals, infusion of leptin induces natriuresis. However, in animal models of enhanced oxidative stress, obesity, or activated renin-angiotensin system, infusion of leptin tends to increase blood pressure. Renin is a critical mediator of angiotensin-II formation, and thus blood pressure control. However, the direct effect of leptin on the release of renin from juxtaglomerular (JG) cells has not been studied. We hypothesize that under normal conditions leptin does not affect renin release whereas during high oxidative stress leptin stimulates renin release. We isolated primary cultures of mouse juxtaglomerular (JG) cells. After treatment with different agonists, renin released to the supernatant was measured by radioimmunoassay. We first tested the direct effect of murine leptin on renin release. We found that 1 hr treatment with leptin (0.1-1 uM) decreased basal renin by 20 ± 5% (p<0.05; n=8). Treatment of JG cells for longer periods (4 and 24 hrs) did not affect renin release (n=6) or total renin expression (n=4). We then tested whether oxidative stress modified the effect of leptin on renin release. For this we pre-incubated JG cells with 10 uM hydrogen peroxide (H2O2) for 1 hr. After this, the medium was removed and H2O2 was completely washed out from JG cells followed by treatment with vehicle (cont) or 10 uM leptin. We found that, in cells pre-treated with H2O2, leptin increased renin release by 49.2 ±16 %(P<0.05 vs vehicle). By Western blot, we detected the expression of the leptin receptor in lysates from JG cells. We concluded that under normal conditions leptin inhibits renin release from JG cells. However, after exposure to H2O2, leptin stimulates renin release. Our data suggest the hypothesis that oxidative stress reverses the inhibitory effect of leptin on renin release and supports a pro-hypertensive role for leptin during chronic inflammatory conditions that induces oxidative stress.

Abstract 085: Role of Suppressor of Cytokines Signaling 3 (Socs3) in Modulating Chronic Metabolic and Cardiovascular Effects of Leptin

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling. Hypothalamic SOCS3 is upregulated in obese animals fed a high-fat diet and has been suggested to contribute to development of resistance to leptin’s anorexic effects. In this study we determined whether deletion of SOCS3 in the entire central nervous system (CNS) amplifies the chronic anorexic and blood pressure (BP) effects of physiological increases in plasma leptin in mice fed a normal diet. SOCS3 flox/flox -Nestin-cre mice were generated by breeding SOCS3 flox/flox with Nestin-cre mice. BP and heart rate (HR) were recorded by telemetry, and oxygen consumption (VO 2 ) was monitored by indirect calorimetry in 22-week-old SOCS3 flox/flox -Nestin-cre (n=4) and control mice (SOCS3 flox/flox , n=4). Compared to controls SOCS3 flox/flox -Nestin-cre mice were lighter (30±1 vs 33±1 g) and normoglycemic (124±7 vs 146±10 mg/dl), consumed less food (3.0±0.4 vs 3.6±0.2 g/day) and had similar VO 2 (77±6 vs 73±3 ml/kg/min). SOCS3 flox/flox -Nestin-cre mice had similar MAP (103±3 vs 107±3 mmHg) but higher HR (666±15 vs 602±17 bpm) compared to control mice. Chronic leptin infusion greatly reduced food in SOCS3 flox/flox -Nestin-cre (46±3 vs 35±4%) and increased MAP (15±3 vs 7±2 mmHg) and VO2 (18±3 vs 14±2%) compared to control mice. No significant changes were observed in HR in either group. Leptin infusion significantly reduced blood glucose levels in both groups (124±7 to 97±7 vs 146±10 to 105±7 mg/dl). These results indicate that SOCS3 deletion in the entire CNS reduces body weight and food intake, and amplifies leptin’s effect on appetite and blood pressure and also suggest the SOCS3 signaling attenuates the chronic actions of leptin on blood pressure as well as appetite regulation even in non-obese mice fed a normal diet. (NHLBI-PO1HL51971, NIGMS P20GM104357 and AHA SDG5680016)

Abstract 132: Leptin-mediated Aldosterone Secretion Causes Hypertension in Obese Females

Obesity causes hypertension (HTN) in males and females. While leptin contributes to obesity-induced HTN by increasing sympathetic activity, in males, it is unknown whether similar mechanisms trigger HTN in obese females. Females secrete 3 to 4 times more leptin than males, but do not exhibit high sympathetic tone with obesity. They however show inappropriately high aldosterone levels that positively correlate with adiposity and blood pressure (BP). Here we hypothesized that leptin induces HTN by increasing aldosterone production in obese females. Hypersensitivity to leptin, in lean mice deficient in protein tyrosine phosphatase 1B (PTP1B) or high leptin levels, in obese Agouti (Ay/a) mice induced HTN (WT: 115±2; KO: 124±2; a/a: 113±1; Ay/a: 128±7mmHg, p<0.05) but did not increase sympathetic control of BP (response to ganglionic blockade). Leptin sensitization and obesity however elevated plasma aldosterone levels and adrenal aldosterone synthase (CYP11B2) expression, in females. Chronic leptin (KO+AA: 115±5; Ay/a+AA: 114±5mmHg) or mineralocorticoid (KO+spiro:111±5; Ay/a+spiro: 121±6mmHg) receptors inhibition restored BP to baseline levels in females PTP1B KO and obese agouti mice. Leptin or leptin receptor deficiency in female ob/ob and db/db mice, abolished obesity-induced increases in adrenal CYP11B2 and plasma aldosterone while chronic leptin infusion in female mice triggered a dose-dependent increase in adrenal CYP11B2 and plasma aldosterone levels. Leptin-mediated aldosterone secretion was independent of changes in plasma angiotensin II, potassium and corticosterone (index of ACTH levels) and preserved in the presence of losartan or α and β-adrenergic receptors antagonists. Stimulation of human adrenocortical cells with leptin dose-dependently increased CYP11B2 expression and aldosterone production. While investigating the interaction between percentage of body fat, leptin and aldosterone levels in young healthy adult Caucasians we reported a positive correlation between adiposity and aldosterone, and between leptin and aldosterone in adult women only. Together these data suggest that leptin directly regulates aldosterone secretion and that leptin induces HTN via aldosterone dependent mechanisms in obese females.

Chronic central leptin infusion modulates the glycemia response to insulin administration in male rats through regulation of hepatic glucose metabolism

Leptin and insulin use overlapping signaling mechanisms to modify hepatic glucose metabolism, which is critical in maintaining normal glycemia. We examined the effect of an increase in central leptin and insulin on hepatic glucose metabolism and its influence on serum glucose levels. Chronic leptin infusion increased serum leptin and reduced hepatic SH-phosphotyrosine phosphatase 1, the association of suppressor of cytokine signaling 3 to the insulin receptor in liver and the rise in glycemia induced by central insulin. Leptin also decreased hepatic phosphoenolpyruvate carboxykinase levels and increased insulin's ability to phosphorylate insulin receptor substrate-1, Akt and glycogen synthase kinase on Ser9 and to stimulate glucose transporter 2 and glycogen levels. Peripheral leptin treatment reproduced some of these changes, but to a lesser extent. Our data indicate that leptin increases the hepatic response to a rise in insulin, suggesting that pharmacological manipulation of leptin targets may be of interest for controlling glycemia.

Reanalysis of parabiosis of obesity mutants in the age of leptin

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

Intracerebroventricular administration of leptin increase physical activity but has no effect on thermogenesis in cold-acclimated rats.

Most small homotherms display low leptin level in response to chronic cold exposure. Cold-induced hypoleptinemia was proved to induce hyperphagia. However, it is still not clear whether hypoleptinemia regulates energy expenditure in cold condition. We try to answer this question in chronic cold-acclimated rats. Results showed that 5-day intracerebroventricular(ICV) infusion of leptin (5 μg/day) had no effects on basal and adaptive thermogenesis and uncoupling protein 1 expression. Physical activity was increased by leptin treatment. We further determined whether ghrelin could reverse the increasing effect of leptin on physical activity. Coadministration of ghrelin (1.2 μg/day) completely reversed the effect of leptin on physical activity. Collectively, this study indicated the regulation of leptin on energy expenditure during cold acclimation may be mainly mediated by physical activity but not by thermogenesis. Our study outlined behavioral role of leptin during the adaptation to cold, which adds some new knowledge to promote our understanding of cold-induced metabolic adaptation.

Differences in mitochondrial DNA inheritance and function align with body conformation in genetically lean and fat sheep.

Body weight and adiposity are determined by the balance between energy intake, energy expenditure, and nutrient deposition. We have identified differences in appetite-regulating peptides in sheep selectively bred to be either lean or fat, wherein gene expression for orexin and melanin-concentrating hormone are elevated in the lean group. Despite this, the underlying mechanisms leading to differences in body composition in the lean and fat lines remains unknown. We measured postprandial temperature in adipose tissue and muscle to ascertain whether a difference in thermogenesis is associated with the difference in body composition in genetically lean (n = 8) and fat (n = 12) ewes. Body weight was higher (P < 0.01) but percent fat mass was lower (P < 0.001) in the lean group. The percent lean mass was similar in lean and fat groups. Animals received intracerebroventricular cannulae and temperature probes implanted into the retroperitoneal fat and the hind-limb skeletal muscle (vastus lateralis). Animals were meal fed (1100-1600 h) to entrain postprandial thermogenesis. Food intake was similar between lean and fat animals. Postprandial thermogenesis was greater (P < 0.05) in the retroperitoneal adipose tissue of lean animals but not in skeletal muscle. Intracerebroventricular infusion of leptin reduced (P< 0.05) food intake by an equal extent in both groups. Postprandial expression of UCP1 mRNA was greater (P < 0.05) in retroperitoneal fat of lean animals, with similar UCP3 expression in skeletal muscle. Mitochondrial genome sequencing indicated haplotypic clustering in lean and fat animals within both the encoding and nonencoding regions. This demonstrates that differences in body composition may be underpinned by differences in thermogenesis, specifically within adipose tissue. Furthermore, thermogenic differences may be associated with specific mitochondrial DNA haplotypes, suggesting a strong genetic component inherited through the maternal lineage.

Leptin in the Hindbrain Facilitates Phosphorylation of STAT3 in the Hypothalamus

Leptin receptors (ObRb) in the forebrain and hindbrain are each independently recognized as important mediators of leptin‐induced changes in energy balance. We recently used sub‐threshold doses of leptin to show that chronic activation of both hypothalamic and brainstem ObRb is required to reduce body fat. The objective of this study was to identify selectively activated brain nuclei in rats receiving chronic infusions of sub‐threshold leptin in both forebrain and hindbrain. Male Sprague Dawley rats were fitted with 3rd and 4th ventricle cannulas. Alzet pumps were used to infuse either saline (S) or leptin (L) (0.1 µg/24h in 3rd, 0.6 µg/24h in 4th) for 6 days, resulting in four treatment groups SS, SL, LS, LL (3th‐4th). There was no effect of leptin in single ventricle infused groups (LS, SL) compared to controls (SS). Double ventricle infused rats (LL) showed a 50% inhibition of food intake, 30% loss in body fat, and an increase in ObRb activation marker phosphorylated STAT3 (pSTAT3‐ir) in the arcuate nucleus (Arc), ventromedial (VMH), dorsomedial (DMH) and posterior hypothalamus compared to other groups. No differences in pSTAT3‐ir were observed in midbrain or brainstem nuclei despite a 6 fold higher infusion of leptin into the 4th ventricle than the 3rd. Delta Fos B‐ir, a chronic neuronal activation marker, showed that multiple brain nuclei were chronically activated due to the process of infusion, but only the Arc, VMH, DMH and ventral tuberomammillary nucleus showed a significant increase in LL rats compared to other groups. These data imply that the simultaneous activation of both forebrain and hindbrain ObRb is required to reduce body fat and that leptin in the hindbrain facilitates phosphorylation of STAT3 in hypothalamic areas involved in the control of energy balance.

Deletion of Protein Tyrosine Phosphatase 1b (Ptp1b) in Proopiomelanocortin (POMC) Neurons Reduces Sympathetic Tone and Protects Mice from Leptin‐Induced High Blood Pressure

Ptp1b is a negative regulator of leptin signaling. Deletion of Ptp1b in POMC neurons increases energy expenditure and protects mice from obesity and type 2 diabetes. However, whether POMC‐Ptp1b deletion sensitizes the cardiovascular system to leptin is unknown. We addressed this question by analyzing the cardiovascular phenotype of flox/flox (WT) and POMC‐Ptp1b KO mice (KO), at baseline and after 7 days of chronic leptin infusion. Conscious blood pressure (BP) and heart rate recordings revealed that POMC‐Ptp1b deletion did not affect baseline BP [WT: 118.3±4.1 vs KO: 117.9± 4.2 mmHg]. However KO mice exhibit a lower BP response to ganglionic blockade [WT: ‐30±4 vs KO: ‐15±6 % drop, P&lt;0.05] that suggests a decreased neurogenic control of BP. Reduced sympathetic tone was further confirmed by showing that KO mice exhibit low plasma catecholamine levels [WT: 3765±483 vs KO: 2113±521 pg/mL, P&lt;0.05, HPLC] compared to WT mice and a compensatory increase in vascular adrenergic tone (increased vascular constriction to phenylephrine) and a1 adrenergic receptors expression (qRT‐PCR). Furthermore chronic sympatho‐activation for 7 days via phenylephrine infusion restored vascular adrenergic tone in KO mice. POMC‐Ptp1b deletion abolished leptin‐induced increase in BP [WT: +13±2.5 mmHg, P&lt;0.05 vs baseline; KO: +4±1.9 mmHg, NS]. However chronic leptin infusion restored vascular adrenergic tone in POMC‐Ptp1b KO mice by increasing vascular sympathoactivation. These data suggests that deletion of Ptp1b in POMC neurons protects from leptin‐induced high BP by reducing neurogenic control of blood pressure.

Leptin: a potential anxiolytic by facilitation of fear extinction.

Anxiety disorders are characterized by a deficient extinction of fear memory. Evidence is growing that leptin influences numerous neuronal functions. The aims of this study were to investigate the effects and the mechanism of leptin on fear extinction. Leptin (1 mg/kg, i.p) was applied to evaluate the anxiolytic effect in rat behavioral tests. Field potentials recording were used to investigate the changes in synaptic transmission in the thalamic-lateral amygadala (LA) pathway of rat. We found that leptin produced strong anxiolytic effects under basal condition and after acute stress. Systemic administration and intra-LA infusions of leptin facilitated extinction of conditioned fear responses. The antagonist of NMDA receptor, MK-801, blocked the effect of leptin on fear extinction completely. Furthermore, these effects of leptin on fear extinction were accompanied by a reversal of conditioning-induced synaptic potentiation in the LA. Leptin facilitated NMDA receptor-mediated synaptic transmission, and reversed amygdala long-term potentiation (LTP) in a dose-dependent manner in vitro, and this LTP depotentiation effect was mediated by NMDA receptor and MAPK signaling pathway. These results identify a key role of leptin in dampening fear conditioning-induced synaptic potentiation in the LA through NMDA receptor and indicate a new strategy for treating anxiety disorders.

Insensitivity of well-conditioned mature sheep to central administration of a leptin receptor antagonist

Ruminants remain productive during the energy insufficiency of late pregnancy or early lactation by evoking metabolic adaptations sparing available energy and nutrients (e.g. higher metabolic efficiency and induction of insulin resistance). A deficit in central leptin signaling triggers these adaptations in rodents but whether it does in ruminants remains unclear. To address this issue, five mature ewes were implanted with intracerebroventricular (ICV) cannula in the third ventricle. They were used in two experiments with an ovine leptin antagonist (OLA) when well-conditioned (average body condition score of 3.7 on a 5 point scale). The first experiment tested the ability of OLA to antagonize leptin under in vivo conditions. Ewes received continuous ICV infusion of artificial cerebrospinal fluid (aCSF), ovine leptin (4 µg/h) or the combination of ovine leptin (4 µg/h) and its mutant version OLA (40 µg/h) for 48 h. Dry matter intake (DMI) was measured every day and blood samples were collected on the last day of infusion. ICV infusion of leptin reduced DMI by 24% (P<0.05), and this effect was completely abolished by OLA co-infusion. A second experiment tested whether a reduction in endogenous leptin signaling in the brain triggers metabolic adaptations. This involved continuous ICV infusions of aCSF or OLA alone (40 µg/h) for 4 consecutive days. The infusion of OLA did not alter voluntary DMI over the treatment period or on any individual day. OLA did not affect plasma variables indicative of insulin action (glucose, non-esterified fatty acids, insulin and the disposition of plasma glucose during an insulin tolerance test) or plasma cortisol, but tended to reduce plasma triiodothyronine and thyroxine (P<0.07). Overall, these data show that a reduction of central leptin signaling has little impact on insulin action in well-conditioned mature sheep. They also raise the possibility that reduced central leptin signaling plays a role in controlling thyroid hormone production.

Leptin in the hindbrain facilitates phosphorylation of STAT3 in the hypothalamus.

Leptin receptors (ObRs) in the forebrain and hindbrain have been independently recognized as important mediators of leptin responses. We recently used low-dose leptin infusions to show that chronic activation of both hypothalamic and hindbrain ObRs is required to reduce body fat. The objective of the present study was to identify the brain nuclei that are selectively activated in rats that received chronic infusion of leptin in both the forebrain and hindbrain. Either saline or leptin was infused into third and fourth ventricles (0.1 μg/24 h in the third ventricle and 0.6 μg/24 h in the fourth ventricle) of male Sprague-Dawley rats for 6 days using Alzet pumps. Rats infused with leptin into both ventricles (LL rats) showed a significant increase in phosphorylated (p)STAT3 immunoreactivity in the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and posterior hypothalamus compared with other groups. No differences in pSTAT3 immunoreactivity were observed in midbrain or hindbrain nuclei despite a sixfold higher infusion of leptin into the fourth ventricle than the third ventricle. ΔFosB immunoreactivity, a marker of chronic neuronal activation, showed that multiple brain nuclei were chronically activated due to the process of infusion, but only the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and ventral tuberomamillary nucleus showed a significant increase in LL rats compared with other groups. These data demonstrate that low-dose leptin in the hindbrain increases pSTAT3 in areas of the hypothalamus known to respond to leptin, supporting the hypothesis that leptin-induced weight loss requires an integrated response from both the hindbrain and forebrain.

Leptin improves fatty liver independently of insulin sensitization and appetite suppression in hepatocyte-specific Pten-deficient mice with insulin hypersensitivity.

Nonalcoholic fatty liver disease (NAFLD) is recognized as the hepatic component of the metabolic syndrome. Although NAFLD is a major cause of cirrhosis and cancer of the liver of unknown cause, no established pharmacological treatment for NAFLD has been established yet. It has been reported that leptin treatment improved fatty liver dramatically as well as insulin resistance and hyperphagia in patients with lipodystrophy. However, it is unclear whether leptin improves fatty liver independently of these metabolic improvements. We investigated the liver effect of leptin independently of insulin sensitization and appetite suppression using hepatocyte-specific Pten-deficient (AlbCrePtenff) mouse, a model of severe fatty liver with insulin hypersensitivity. Male AlbCrePtenff mice were infused subcutaneously with leptin (20 ng/g/h) for 2 weeks using osmotic minipumps. Leptin infusion effectively reduced liver weight, liver triglyceride content, and glutamate pyruvate transaminase (GPT) concentrations as well as food intake and body weight without the change of plasma insulin concentration in AlbCrePtenff mice. Pair-feeding also reduced body weight but not liver triglyceride content. Pair feeding reduced α1 and α2 AMP-activated protein kinase (AMPK) activities and PGC1α gene expression in the liver, while leptin infusion unchanged them. The present study clearly demonstrated that leptin improve fatty liver independently of insulin sensitization and suppression of food intake. It was suggested that leptin improves fatty liver by stimulation of β-oxidation in the liver. The present study might provide a further understanding on the mechanism of metabolic effect of leptin.

Shp2 signaling in POMC neurons is important for leptin's actions on blood pressure, energy balance, and glucose regulation.

Previous studies showed that Src homology-2 tyrosine phosphatase (Shp2) is an important regulator of body weight. In this study, we examined the impact of Shp2 deficiency specifically in proopiomelanocortin (POMC) neurons on metabolic and cardiovascular function and on chronic blood pressure (BP) and metabolic responses to leptin. Mice with Shp2 deleted in POMC neurons (Shp2/Pomc-cre) and control mice (Shp2(flox/flox)) were implanted with telemetry probes and venous catheters for measurement of mean arterial pressure (MAP) and leptin infusion. After at least 5 days of stable control measurements, mice received leptin infusion (2 μg·kg(-1)·day(-1) iv) for 7 days. Compared with Shp2(flox/flox) controls, Shp2/Pomc-cre mice at 22 wk of age were slightly heavier (34 ± 1 vs. 31 ± 1 g) but consumed a similar amount of food (3.9 ± 0.3 vs. 3.8 ± 0.2 g/day). Leptin infusion reduced food intake in Shp2(flox/flox) mice (2.6 ± 0.5 g) and Shp2/Pomc-cre mice (3.2 ± 0.3 g). Despite decreasing food intake, leptin infusion increased MAP in control mice, whereas no significant change in MAP was observed in Shp2/Pomc-cre mice. Leptin infusion also decreased plasma glucose and insulin levels in controls (12 ± 1 to 6 ± 1 μU/ml and 142 ± 12 to 81 ± 8 mg/100 ml) but not in Shp2/Pomc-cre mice. Leptin increased V̇o2 by 16 ± 2% in controls and 7 ± 1% in Shp2/Pomc-cre mice. These results indicate that Shp2 signaling in POMC neurons contributes to the long-term BP and antidiabetic actions of leptin and may play a modest role in normal regulation of body weight.

An acute non‐lethal method to test leptin responsiveness in rats (371.3)

Continuous subcutaneous administration of leptin normalizes blood glucose levels in rodent models of type I and type II diabetes independent of changes in food intake, body weight and plasma insulin. We tested whether an acute intravenous leptin infusion changed blood glucose in normal and diet‐induced leptin resistant rats and determined if this could be used as a marker of leptin sensitivity. Leptin responsive, chow fed male Sprague Dawley rats and leptin resistant rats fed chow, lard and 30% sucrose or chow and 30% sucrose were fitted with jugular vein catheters. They were infused with either saline for 32 min, low dose (LD) leptin (1.9µg/kg/min) followed by high dose (HD) leptin (3.75µg/kg/min) for 16 min each, or only HD leptin for 16 min. Infusion of either LD leptin or HD leptin alone did not change blood glucose. Infusion of LD leptin followed by HD leptin decreased blood glucose in leptin responsive, but not leptin resistant rats. Leptin infusion increased serum leptin in all groups, but had no effect on serum insulin, glucagon, 18 hour weight gain or 18 hour energy intake in any rat. The leptin‐induced decrease in blood glucose correlated with leptin responsiveness measured as a leptin‐induced inhibition of food intake. This effect is consistent in control rats and absent in two rat models of leptin resistance. Thus, this acute non‐lethal test is a novel method of testing leptin responsiveness in rats.Grant Funding Source: Supported by NIH DK 53903

In vivo and in vitro evidence that leptin stimulates the hexosamine biosynthetic pathway (640.9)

We previously reported that glucosamine administration, which activates the hexosamine biosynthetic pathway (HBP), causes leptin resistance in rats. Here we tested whether nutrient activation of the HBP results in leptin resistance. Male Sprague‐Dawley rats received intraperitoneal infusions of saline or leptin (40 μg/day) for 12 days and were fed chow or a choice diet of chow, 30% sucrose solution, and lard. The choice diet caused leptin resistance and increased hepatic glutamine fructose 6‐phosphate amidotransferase (GFAT), a rate limiting enzyme in the HBP, and O‐linked N‐acetylglucosamine (O‐GlcNAc) protein modification. Leptin infusion increased GFAT and O‐GlcNAcylation in chow‐fed rats, whereas it had no additive effect in choice rats. To test for direct effects of leptin and glucose, HepG2 cells were incubated with high concentrations of glucose (18.5 mM) or with 5.5 mM glucose and leptin (0, 5, 10, 50, 100 or 500 ng/mL). Both leptin and glucose stimulated cellular GFAT and protein O‐GlcNAcylation. Thus, leptin stimulates the HBP both in vivo and in vitro, and activation of the HBP due to increased nutrient flux is associated with leptin resistance.Grant Funding Source: Supported by NIH grant DK 53903

Central leptin sensitization of angiotensin II‐induced hypertension (858.3)

Obesity has been associated with selective leptin resistance whereby the anorexic effect of leptin is attenuated, but its action to increase sympathetic nervous activity (SNA) and blood pressure (BP) remains intact. A recent study showed an interaction between leptin and the renin angiotensin system in regulating SNA. Our previous studies using an Induction‐Delay‐Expression experimental design have demonstrated that a subpressor dose of angiotensin (ANG) II pretreatment sensitizes the brain to produce an enhanced hypertensive response to a subsequent treatment with pressor dose of ANG II. The present study tested the hypothesis that central chronic leptin pretreatment can also sensitize the pressor actions of ANG II. Male rats were implanted for telemetered BP recording and were pretreated during Induction with either peripheral subpressor ANG II (10 ng/kg/min) or central leptin (20 ng/kg/min) for one week. After one week Delay, rats were treated during Expression with subcutaneous ANG II (120 ng/kg/min) for 2 weeks. The animals with the subpressor ANG II pretreatment responded with enhanced hypertension to ANG II (Δ44.5±3.4 mmHg vs.Δ 21.2±5.2 mmHg). Central infusion of leptin during Induction mimicked the sensitization produced by the subpressor ANG II (Δ39.3±3.8 mmHg). The results indicate that central chronic leptin can predispose rats to display an enhanced ANG II‐elicited hypertensive response.