Injection Of Leptin Research Articles

Leptin receptor-expressing cells in the ventromedial nucleus of the hypothalamus contribute to enhanced CCK-induced satiety following central leptin injection.

Others have shown that leptin and cholecystokinin (CCK) act synergistically to suppress food intake. Experiments described here tested whether leptin in the ventromedial hypothalamus (VMH) contributes to the synergy with peripheral CCK in male Sprague Dawley rats. A subthreshold injection of 50-ng leptin into the VMH 1 h before a peripheral injection of 1 µg/kg CCK did not change the response to CCK in rats offered chow or low-fat purified diet, but did exaggerate the reduction in intake of high-fat diet 30 min and 1 h after injection in rats that had been food deprived for 8 h. By contrast, deletion of leptin receptor-expressing cells in the VMH using leptin-conjugated saporin (Lep-Sap) abolished the response to peripheral CCK in chow-fed rats. Lateral ventricle injection of 2-µg leptin combined with peripheral CCK exaggerated the inhibition of chow intake for up to 6 h in control rats treated with Blank-saporin, but not in Lep-Sap rats. Blank-Saporin rats offered low- or high-fat purified diet also demonstrated a dose-response inhibition of intake that reached significance with 1 µg/kg of CCK for both diets. CCK did not inhibit intake of Lep-Sap rats in either low- or high-fat-fed rats. Thus, although basal activation of VMH leptin receptors makes a significant contribution to the synergy with CCK, increased leptin activity in the VMH does not exaggerate the response to CCK in intact rats offered low-fat diets, but does enhance the response in those offered high-fat diet.NEW & NOTEWORTHY Leptin is a feedback signal in the control of energy balance, whereas cholecystokinin (CCK) is a short-term satiety signal that inhibits meal size. The two hormones synergize to promote satiety. We tested whether leptin receptors in the ventromedial nucleus of the hypothalamus (VMH) contribute to the synergy. The results suggest that there is a requirement for a baseline level of activation of leptin receptors in the VMH in order for CCK to promote satiety.

Influence of In Ovo Leptin Injection into Yolk Sac on Embryo Development, Blood Biochemical Parameters and Lipid Metabolism of Broiler Chicks during Early Post-Hatching Period

The study aimed to investigate the effect of in ovo (IO) leptin injection on yolk sac leptin concentration, embryo development, blood lipid metabolism, and biochemical parameters of broiler chicks during the early post-hatching period. Hatching eggs were weighed and placed in an incubator with 100 eggs/4 replications/group. The groups were: non-injected group (C); and 3 injected groups with 100 μl of phosphate buffer solution (PBS); 0.5 μl leptin (L0.5), and 1 μl leptin (L1) in 100 μl of PBS. Pure leptin hormone dissolved in PBS was injected into the yolk sac on day 7 of incubation. Yolk/yolk sac leptin concentrations at the onset of incubation, on days 7 and 15 of incubation, and at hatching were determined. Embryo/chick development was determined at all ages. Serum leptin, tri-iodothyronine (T3), thyroxine (T4), creatine kinase, total protein, uric acid, and lipid profiles were measured at hatching and day 7 of chick age. The leptin level of the yolk was similar to the leptin level of the yolk sac at hatching. Leptin level of a yolk sac was higher on 7th day than at other ages. IO injection* age was significant for serum leptin level, which was similar to one-day chicks in the L0.5 at day 7 of chick age. T3, T4, creatine kinase, uric acid, lipid profiles, and embryo/chick development did not change with IO leptin. Serum HDL level was higher in the leptin groups than C and PBS groups at hatching. IO leptin application affects blood lipid metabolism depending on dose level for male broiler chicks at the early post-hatch period, without affecting embryo/chick development.Leptin level of yolk was similar to leptin level of yolk sac at hatching. Leptin level of yolk sac was higher on 7th day than other ages. IO injection* age was significant for serum leptin level, which was similar to one-day chicks in the L0.5 at day 7 of chick age. T3, T4, creatine kinase, uric acid, lipid profiles, and embryo/chick development did not change with IO leptin. Serum HDL level was higher in the leptin groups than C and PBS groups at hatching. IO leptin application affects blood lipid metabolism depending on dose level for male broiler chicks at early post-hatch period, without affecting embryo/chick development.

Influence of Leptin on Severity of Acute Pancreatitis in Experimental Rat Model

Background and Aim: In acute pancreatitis, hyperproduction of inflammatory mediators, including inflammatory cytokines, exacerbation of tissue injury due to microcirculatory disturbances, secondary activation of inflammatory cells in distant organs, and the cascade are the pathogenic basis of pancreatitis severity. Therefore, inhibition of pancreatic enzyme activity, blocking production, and preventing the activation of inflammatory cells is a practical way to prevent the development of severe acute pancreatitis and multiorgan failure. In our study, we sought to elucidate the effect of leptin on a rat model of severe acute pancreatitis. Methods: We prepared an acute pancreatitis model in rats by intraperitoneal injection of 20μg/kg of leptin and single injection of 0.1mL/100g of bile into a pancreatic duct extending from the duodenal papilla, measured survival, serum amylase activity, MPO activity in pancreatic tissue, SOD activity and MDA content, and observed the histopathological findings of pancreatic tissue. Results: Pre-administration of 20μg/kg leptin improved the survival of rats, decreased serum amylase activity, MPO activity in pancreatic tissue, increased MDA content, increased SOD activity, and improved the histopathological features of pancreatic tissue. Conclusion: Leptin administration prevented the destruction of pancreatic cells and improved the antioxidant function in rats with severe acute pancreatitis.

Altered ERK‐mediated control of AgRP and metabolic rate during obesity

Adaptation of resting metabolic rate (RMR) contributes to the maintenance of human obesity, yet RMR control and how this system changes during obesity remains poorly defined. Desensitization of the brain to leptin has been proposed to contribute. Leptin normally suppresses Agouti‐related peptide (AgRP) expression in the arcuate nucleus of the hypothalamus (ARC) to disinhibit (increase) melanocortin‐mediated RMR stimulation. Diet‐induced obesity (DIO) increases leptin, but paradoxically exaggerates Agrp expression and causes RMR adaptation. Thus, something connecting leptin to Agrp control becomes dysfunctional during DIO. Our previous data indicate that within the ARC, disruption in the extracellular signal‐regulated kinase (ERK) cascade occurs during DIO. ERK normally phosphorylates Krüppel‐like factor 4 (KLF4), inactivating it. As KLF4 binds to the AgRP promoter to increase transcription, its inactivation results in reduced Agrp expression. We hypothesize that this mechanism becomes dysfunctional within AgRP neurons during DIO, such that KLF4 binding to the AgRP promoter increases despite increased ERK activity.Mouse hypothalamic GT1‐7 cells (model of AgRP neurons) were treated with leptin (1 uM, 15 min or 1 hr) and pERK/ERK ratio was measured by Simple WES and Agrp mRNA expression via qPCR, respectively. Leptin was given to chow‐fed adult mice (2 ug/g ip 30 min) and ARC pERK/ERK ratio was measured, as well as RMR assessed by respirometry. To examine consequences of ERK or KLF4 disruption upon cardiometabolic phenotypes, mice harboring a conditional version of endogenous Mapk1 or Klf4 were crossed with mice expressing Cre via the Agrp locus.In GT1‐7 cells, leptin increased pERK/ERK ratio (1 uM, 15 min leptin n=3, +89% vs vehicle n=2, p<0.05) and suppressed Agrp expression (1 uM, 1 hr leptin n=2, ‐54.1% vs vehicle n=6, p<0.05). In chow‐fed adult mice, leptin injection caused an increase in the ARC pERK/ERK ratio (2 ug/g ip 30 min leptin n=11, +43.8% vs saline n=9, p<0.05). Additionally, leptin stimulated RMR in adult‐chow fed mice (1 ug/g ip leptin n=8, 16.5‐fold of vehicle n=8, p<0.05). DIO caused increases in endogenous plasma leptin and an increase in the ARC pERK/ERK ratio (High Fat Diet (HFD; n=5), +54.4% vs chow n=4, p<0.05), but ARC Agrp expression was not suppressed (HFD n=4, 1.62 fold of chow n=4, p<0.05). There was a possible exaggeration of weight gain in Agrp‐Cre+, Mapk1F/F mice compared to controls following HFD (KOs n=2, +16.8% vs controls n=3). In contrast, weight gain was possibly attenuated in Agrp‐Cre+, Klf4F/F mice compared to controls following HFD (KO n=1, ‐10.7% vs controls n=2). RMR was higher in chow‐fed Agrp‐Cre+, Klf4F/F mice compared to controls (KOs n=5 +8.3% vs controls n=7, p<0.05) and may be exaggerated following HFD (KO n=1 +13.0% vs controls n=3). Together, these findings suggest that ERK and KLF4 within AgRP neurons appear to contribute to the control of AgRP and thus RMR. These findings may identify ERK control of KLF4 and KLF4 as a potential therapeutic target to maintain/restore RMR control after prolonged obesity.

Role of Hypothalamic Serotonin Receptors in Controlling Food Intake Following Pharmacological Administration of Leptin

Use of leptin as a pharmacological intervention for obesity has failed since its discovery in 1994. This failure is attributed to the already high levels of leptin circulating in most obese patients, contributing to the lack of response from the brain, termed “leptin resistance”. Still there is hope that leptin may yet be used as a treatment for obesity. Leptin receptors are expressed throughout the brain, showing that the anorectic function of leptin does not rely solely on direct stimulation of hypothalamic neurons. In our laboratory, using pharmacological and optogenetic approaches we have demonstrated that food intake is also controlled by the leptin receptors located in the dorsal raphe nuclei (DRN). We have also utilized retrograde tracing to characterize a neural pathway from the DRN to the arcuate of the hypothalamus that is dependent on leptin. Then, through pharmacological suppression of serotonin (5HT) production, we showed that an intact pool of 5HT in the DRN is necessary for leptin to reduce food intake. Therefore, our objective for this study is to understand the role of specific serotonin receptors in the anorectic effects of leptin through the raphe. We hypothesize that both the 2C and 1B 5HT receptors are necessary for the anorectic effects of leptin through the raphe, and only blocking one will show partial decrease of leptin’s effects. To address this hypothesis, we performed stereotaxic surgery on adult male Sprague Dawley rats to implant cannulas into the third ventricle in the vicinity of hypothalamic nuclei. Following recovery, they were habituated to metabolic cages for 7 days. Then, we administered selective antagonists for the 5HT2C (SB242084) and/or 5HT1B (GR55562) receptors (or vehicle as control) into the third ventricle to block these receptors throughout the hypothalamus. Thirty minutes later, we administered leptin to the DRN, done just before the onset of the dark cycle. For 24 hours, the metabolic cages automatically measured food intake. Five days after the administration, we reversed groups, giving vehicle to the antagonist treated groups and vice versa, recording food intake in the same way. Five days after the second administration, we injected antagonist or vehicle into the third ventricle, then leptin into the DRN 30 minutes after. Two hours post leptin injection, rats were perfused and fixed with 4% paraformaldehyde and brains were collected. We then sectioned and performed immunofluorescence to label cFOS, a marker of neuron activation, in the hypothalamus. CFos was counted to determine differences between animals treated with antagonist vs vehicle. We observed that food intake was reduced by leptin just as we’ve shown previously. However, this anorectic effect is lessened when the 5HT1B or 5HT2C receptor is given into the third ventricle prior to leptin infusion, but not completely removed. These results strongly suggest that the anorectic effects of leptin and serotonin are strongly intertwined, and understanding this interaction may lead to better pharmacological treatments for obesity and eating disorders.

Spexin Regulates Hypothalamic Leptin Action on Feeding Behavior.

Spexin (SPX) is a recently identified neuropeptide that is believed to play an important role in the regulation of energy homeostasis. Here, we describe a mediating function of SPX in hypothalamic leptin action. Intracerebroventricular (icv) SPX administration induced a decrease in food intake and body weight gain. SPX was found to be expressed in cells expressing leptin receptor ObRb in the mouse hypothalamus. In line with this finding, icv leptin injection increased SPX mRNA in the ObRb-positive cells of the hypothalamus, which was blocked by treatment with a STAT3 inhibitor. Leptin also increased STAT3 binding to the SPX promoter, as measured by chromatin immunoprecipitation assays. In vivo blockade of hypothalamic SPX biosynthesis with an antisense oligodeoxynucleotide (AS ODN) resulted in a diminished leptin effect on food intake and body weight. AS ODN reversed leptin’s effect on the proopiomelanocortin (POMC) mRNA expression and, moreover, decreased leptin-induced STAT3 binding to the POMC promoter sequence. These results suggest that SPX is involved in leptin’s action on POMC gene expression in the hypothalamus and impacts the anorexigenic effects of leptin.

Leptin: A Potential Link Between Obstructive Sleep Apnea and Obesity.

Chronic intermittent hypoxia (CIH), a pathophysiological manifestation of obstructive sleep apnea (OSA), is strongly correlated with obesity, as patients with the disease experience weight gain while exhibiting elevated plasma levels of leptin. This study was done to determine whether a relationship may exist between CIH and obesity, and body energy balance and leptin signaling during CIH. Sprague-Dawley rats were exposed to 96 days of CIH or normoxic control conditions, and were assessed for measures of body weight, food and water intake, and food conversion efficiency. At the completion of the study leptin sensitivity, locomotor activity, fat pad mass and plasma leptin levels were determined within each group. Additionally, the hypothalamic arcuate nucleus (ARC) was isolated and assessed for changes in the expression of proteins associated with leptin receptor signaling. CIH animals were found to have reduced locomotor activity and food conversion efficiency. Additionally, the CIH group had increased food and water intake over the study period and had a higher body weight compared to normoxic controls at the end of the study. Basal plasma concentrations of leptin were significantly elevated in CIH exposed animals. To test whether a resistance to leptin may have occurred in the CIH animals due to the elevated plasma levels of leptin, an acute exogenous (ip) leptin (0.04 mg/kg carrier-free recombinant rat leptin) injection was administered to the normoxic and CIH exposed animals. Leptin injections into the normoxic controls reduced their food intake, whereas CIH animals did not alter their food intake compared to vehicle injected CIH animals. Within ARC, CIH animals had reduced protein expression of the short form of the obese (leptin) receptor (isoform OBR100) and showed a trend toward an elevated protein expression of the long form of obese (leptin) receptor (OBRb). In addition, pro-opiomelanocortin (POMC) protein expression was reduced, but increased expression of the phosphorylated extracellular-signal-regulated kinase 1/2 (pERK1/2) and of the suppressor of cytokine signaling 3 (SOCS3) proteins was observed in the CIH group, with little change in phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Taken together, these data suggest that long-term exposure to CIH, as seen in obstructive sleep apnea, may contribute to a state of leptin resistance promoting an increase in body weight.

Short term effects of restricted food availability and peripheral leptin injections in redheaded bunting, Emberiza bruniceps

Migratory birds need continued food supply and efficient metabolic machinery to meet high energy demands of the magnanimous feat of flight. Two questions are important i.e. as to 1) how a bird adapts to a temporary food constrain on a daily basis, and 2) how peripheral leptin, an anorectic hormone, impacted feeding and migratory behaviour in buntings? The aim of this study was to induce a non-photoperiodic tweak in the physiology of redheaded buntings through exogenous leptin administration and study its effect on their food intake and migratory behaviour. Groups of male redheaded buntings, Emberiza bruniceps (n=17) were transferred from short (8L: 16D) to long (16L: 8D) days and presented with food only either for first (morning food presence, MFP) or second (evening food presence, EFP) half of the 16h lighted phase, while control group received food ad libitum. Total daily food intake (FI) did not differ significantly between the MFP, EFP and controls, but hourly FI in MFP and EFP indicated increased activity differences based on time of food availability and bird’s tendency to cache food/ recompense for food scarcity during migration. In another experiment, a chemical tweak in bird’s FI was induced by peripheral administration of leptin, to add to current understanding of transition in buntings’ metabolic efficiency during high energy demanding migratory journey. Exogenous leptin appeared to safeguard cadaveric effect of exogenous injection in migrating buntings through promoting blood cholesterol and reduced liver fibrosis. Food restriction in the morning was better responded by buntings than that in evening. Therefore, migratory buntings exhibited diurnal variation in response to food scarcity.

Evaluation of the Central Effects of Systemic Lentiviral-Mediated Leptin Delivery in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes (T1D) is characterized by hyperphagia, hyperglycemia and activation of the hypothalamic–pituitary–adrenal (HPA) axis. We have reported previously that daily leptin injections help to alleviate these symptoms. Therefore, we hypothesized that leptin gene therapy could help to normalize the neuroendocrine dysfunction seen in T1D. Adult male Sprague Dawley rats were injected i.v. with a lentiviral vector containing the leptin gene or green fluorescent protein. Ten days later, they were injected with the vehicle or streptozotocin (STZ). HPA function was assessed by measuring norepinephrine (NE) levels in the paraventricular nucleus (PVN) and serum corticosterone (CS). Treatment with the leptin lentiviral vector (Lepvv) increased leptin and insulin levels in non-diabetic rats, but not in diabetic animals. There was a significant reduction in blood glucose levels in diabetic rats due to Lepvv treatment. Both NE levels in the PVN and serum CS were reduced in diabetic rats treated with Lepvv. Results from this study provide evidence that leptin gene therapy in STZ-induced diabetic rats was able to partially normalize some of the neuroendocrine abnormalities, but studies with higher doses of the Lepvv are needed to develop this into a viable option for treating T1D.

Impact of the Central Histaminergic and Melanocortin Systems on Leptin-Induced Hypophagia in Neonatal Layer Chicken.

The present study aimed to assess the probable impact of the central histaminergic and melanocortin systems on leptin-induced hypophagia in neonatal layer chickens. In experiment 1, the chickens received intracerebroventricular (ICV) injections of the control solution, 250 nmol of α-FMH, 10 µg of leptin, and α-FMH+leptin. Experimental groups 2-8 were injected the same as experiment 1. Nonetheless, the chickens in experiments 2-8 received ICV injections of 300 nmol of chlorpheniramine (H1 receptor antagonist), 82 nmol of famotidine (H2 receptor antagonist), 300 nmol of thioperamide (H3 receptor antagonist), 0.5 nmol of SHU9119 (M3/M4 receptors antagonist), 0.5 nmol of MCL0020 (M4 receptor antagonist), 30 µg of astressin-B (CRF1/ CRF2 receptors antagonist), and 30 µg of astressin2-B (CRF2 receptor antagonist), instead of α-FMH, respectively. Food was provided for the birds immediately following the injection, and 30, 60, and 120 min after the injection, cumulative food intake (g) was measured. The findings pointed out that the ICV injection of leptin diminished food intake in neonatal chickens (P<0.05). The co-administration of M3/M4 receptor antagonist+leptin significantly decreased the hypophagic effect of leptin (P<0.05). A significant decrease was also detected in the hypophagic effect of leptin following the co-administration of the M4 receptor antagonist and leptin (P<0.05). Moreover, the co-injection of the antagonists of CRF1/CRF2 receptors and leptin significantly mitigated the hypophagic effect of leptin (P<0.05). The co-injection of CRF2 receptor antagonist and leptin led to a decrease in the hypophagic effect of leptin. As evidenced by the results of the current study the hypophagic effect of leptin is mediated by the receptors of H1, H3, M3/M4, and CRF1/CRF2 in neonatal layer chicken.

Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

Background: Polycystic ovary syndrome (PCOS) is a common reproductive and metabolic disorder characterized by high androgen levels. The aim of this study was to evaluate the effects of hyperandrogenism on the hypothalamus and subsequently on the food intake and obesity in females. Methods: A dihydroxy testosterone (DHT)-induced rat model was established to recapitulate the hyperandrogenism features of PCOS patients. Body weight and food intake of the rats were recorded. The food intake of DHT-induced rats was restricted by pair feeding to exclude possible effects of weight gain on the hypothalamus. The expression levels of relevant proteins and mRNAs in the hypothalamus and primary hypothalamic neurons exposed to DHT were analyzed by Western blotting and RT-PCR, respectively. The leptin levels in the serum and cerebrospinal fluid (CSF) were measured, and leptin was injected via the intracerebroventricular (ICV) route to test the leptin sensitivity of the hypothalamus. Results: The excessive prepuberty androgen levels in the DHT-induced rats markedly elevated food intake prior to weight gain. Consistent with this, the expression of neuropeptide Y and agouti-related peptide mRNAs was upregulated, which occurred prior to obesity and even with restricted food intake. In addition, the hypothalamic sensitivity to insulin and leptin was also impaired in the DHT-induced rats before obesity and with restricted food intake. DHT significantly reduced the leptin levels in the CSF, and ICV injection of leptin inhibited the DHT-induced increase in food intake. Conclusions: Androgen excess increased food intake in rats and promoted obesity by downregulating insulin and leptin signaling in the hypothalamus, most likely by suppressing leptin levels in the CSF.

Undernutrition and Hypoleptinemia Modulate Alloimmunity and CMV-specific Viral Immunity in Transplantation.

Immunological mechanisms linking undernutrition to infection and the alloimmune response are poorly understood in transplantation. We aimed to determine how undernutrition and hypoleptinemia impact T-cell allospecific and cytomegalovirus (CMV) viral-specific immunity in a murine model. Fed, fasted for 48 h (model of undernutrition), and fasted with leptin injections (leptin rescue), C57BL/6 mice received skin grafts from either C57BL/6 (syngeneic) or BALB/c (allogeneic) mice donors. Allograft rejection and survival were monitored. Fed, fasted, and leptin rescue C57BL/6 mice were inoculated with murine cytomegalovirus (mCMV). Mouse spleens were retrieved for T-cell flow cytometry analysis, mCMV DNA extraction, and quantitative polymerase chain reaction. Serum leptin levels were measured with ELISA. Fasted mice had prolonged rejection-free and graft survival compared with fed mice (P = 0.0002 and P = 0.043). Leptin administration did not alter rejection-free survival or allograft failure. CD8+ central memory T cell and CD8+ effector T cell proportions were significantly lower in fasted mice receiving allogeneic skin transplants compared with fed mice (P = 0.0009 and P = 0.0015). Fasted mice had higher viral loads (P = 0.0028) and impaired mCMV-specific interferon-gamma-producing CD8+ T cells (P = 0.0007), which improved with leptin rescue (P = 0.032). Undernutrition and its associated hypoleptinemia correlated with impaired allospecific and viral-specific immunities. Leptin administration decreased mCMV viral burden and increased mCMV-specific T-cell immunity, however, it did not increase rejection or worsen graft survival in complete major histocompatibility complex-mismatched skin allografts. Leptin may be a potential adjunctive therapy for CMV viremia in undernourished transplant recipients.

Abstract P157: Altered ERK-mediated Control Of AgRP And Metabolic Rate During Obesity

Adaptation of resting metabolic rate (RMR) contributes to the maintenance of human obesity, yet the control of RMR - and how this system changes during obesity - remains poorly defined. Leptin normally suppresses Agouti-related peptide (AgRP) expression in the arcuate nucleus of the hypothalamus (ARC) to disinhibit melanocortin-mediated stimulation of RMR. Leptin stimulates ERK signaling in the ARC (a known suppressor of AgRP), and the inhibition of ARC ERK attenuates thermogenic sympathetic nervous activity responses to leptin. Diet-induced obesity (DIO) increases leptin, but paradoxically exaggerates ARC AgRP expression and causes RMR adaptation. Thus, we hypothesize that leptin increases ERK signaling in AgRP cells, to reduce AgRP and thereby ultimately increase RMR, but that this signaling mechanism becomes dysfunctional during DIO. Leptin application to GT1-7 cells (a model of AgRP neurons) increased pERK/ERK ratio (1 uM, 15 min leptin n=3, 2.9-fold of vehicle n=2, p&lt;0.05). In chow-fed adult mice, leptin injection caused an increase in ARC pERK/ERK ratio (2 ug/g ip 30 min leptin n=11, +43.8% vs saline n=9, p&lt;0.05) without affecting total ERK levels (normalized to beta-actin). DIO caused increases in endogenous plasma leptin and an increase in ARC pERK/ERK ratio (HFD n=5, +54.4% vs chow n=4, p&lt;0.05) without affecting total ERK levels - yet, canonical transcriptomic effects of ERK (ie, suppression of AgRP) were lost. Finally, reanalysis of our published single-nucleus RNAsequencing dataset describing the transcriptome of ARC after DIO demonstrates loss of canonical effects of increased ERK signaling specifically within the AgRP cell type. Together, these findings indicate that although DIO is associated with expected increases in ERK activation in AgRP neurons and the ARC, the consequences of ERK signaling are attenuated. We therefore hypothesize that DIO results in RMR adaptation through changes in signaling mechanisms that link ERK to its downstream targets, such as AgRP. Ongoing studies are focused on the development of novel animal models with tamoxifen-dependent conditional deletion of ERK within the adult AgRP neuron, to test whether loss of ERK signaling is sufficient to explain changes in ERK/AgRP/RMR control during DIO.

Activating thermogenesis to combat obesity: hot topic or cold front?

Activating thermogenesis to combat obesity: hot topic or cold front?

Modulation of Sirt1 and FoxO1 on Hypothalamic Leptin‐Mediated Sympathetic Activation and Inflammation in Diet‐Induced Obese Rats

BackgroundHypothalamic leptin‐mediated signaling contributes to the exaggerated sympatho‐excitation and increased blood pressure in obesity‐associated hypertension. The aim of the study was to investigate the roles of energy‐sensing enzyme sirtuin1 (Sirt1) and forkhead box protein O1 (FoxO1) on the hypothalamic leptin‐mediated high sympathetic nerve activity and inflammation in obesity.Methods and ResultsSprague Dawley rats were fed with high‐fat diet (HFD) for 12 weeks. In vivo, the potential of Srit1 and FoxO1 in the sympathetic effects of leptin was investigated via siRNA injection to knockdown Sirt1 or FoxO1 gene in the arcuate nucleus (ARCN) of hypothalamus in rats. In vitro, the effects of Sirt1 or FoxO1 on leptin‐mediated inflammation were observed in proopiomelanocortin (POMC) and microglial cells. Knockdown Sirt1 by siRNA significantly reduced the renal sympathetic nerve activity (RSNA) and blood pressure responses to leptin injection in the ARCN in the HFD rats. Conversely, knockdown FoxO1 significantly enhanced the RSNA and blood pressure responses to leptin injection in the HFD rats. Knockdown Sirt1 reduced the levels of pro‐inflammatory cytokines interleukin 6 (IL‐6), tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), C1q/TNF‐related protein‐1 (CTRP1), and immune cell infiltration in the ARCN in the HFD rats. Knockdown FoxO1 significantly increased the level of IL‐6 in the ARCN of HFD rats. In cultured hypothalamic POMC and microglial cells, knockdown Sirt1 significantly reduced leptin‐induced IL‐6 expression, affected the levels of AMP‐activated protein kinase (AMPK) and serine/threonine‐specific protein kinase (Akt). Knockdown FoxO1 significantly increased leptin‐induced IL‐6 in both POMC cells and microglial cells.ConclusionsThese data suggest that both Sirt1 and FoxO1 are the key modulators of leptin signaling in the hypothalamus contributed to the over sympathetic activation and inflammation in obesity.

Characterization of the onset of leptin effects on the regulation of energy balance.

Leptin is a hormone required for the regulation of body weight in adult animals. However, during the postnatal period, leptin is mostly involved in developmental processes. Because the precise moment at which leptin starts to exert its metabolic effects is not well characterized, our objective was to identify the approximate onset of leptin effects on the regulation of energy balance. We observed that male Lepob/ob mice started to exhibit increased body fat mass from postnatal day 13 (P13), whereas in females, the increase in adiposity began on P20. Daily leptin injections from P10 to P22 did not reduce the weight gain of WT mice. However, an acute leptin injection induced an anorexigenic response in 10-day-old C57BL/6 mice but not in 7-day-old mice. An age-dependent increase in the number of leptin receptor-expressing neurons and leptin-induced pSTAT3 cells was observed in the hypothalamus of P7, P10 and P16 mice. Leptin deficiency started to modulate the hypothalamic expression of transcripts involved in the regulation of metabolism between P7 and P12. Additionally, fasting-induced hypothalamic responses were prevented by leptin replacement in 10-day-old mice. Finally, 12-day-old males and females showed similar developmental timing of axonal projections of arcuate nucleus neurons in both WT and Lepob/ob mice. In summary, we provided a detailed characterization of the onset of leptin's effects on the regulation of energy balance. These findings contribute to the understanding of leptin functions during development.

BACE2 suppression in mice aggravates the adverse metabolic consequences of an obesogenic diet

ObjectivePancreatic β-cell dysfunction is a central feature in the pathogenesis of type 2 diabetes (T2D). Accumulating evidence indicates that β-site APP-cleaving enzyme 2 (BACE2) inhibition exerts a beneficial effect on β-cells in different models of T2D. Thus, targeting BACE2 may represent a potential therapeutic strategy for the treatment of this disease. Here, we aimed to investigate the effects of BACE2 suppression on glucose homeostasis in a model of diet-induced obesity. MethodsBACE2 knock-out (BKO) and wild-type (WT) mice were fed with a high-fat diet (HFD) for 2 or 16 weeks. Body weight, food intake, respiratory exchange ratio, locomotor activity, and energy expenditure were determined. Glucose homeostasis was evaluated by glucose and insulin tolerance tests. β-cell proliferation was assessed by Ki67-positive nuclei, and β-cell function was determined by measuring glucose-stimulated insulin secretion. Leptin sensitivity was evaluated by quantifying food intake and body weight after an intraperitoneal leptin injection. Neuropeptide gene expression and insulin signaling in the mediobasal hypothalamus were determined by qPCR and Akt phosphorylation, respectively. ResultsAfter 16 weeks of HFD feeding, BKO mice exhibited an exacerbated body weight gain and hyperphagia, in comparison to WT littermates. Glucose tolerance was similar in both groups, whereas HFD-induced hyperinsulinemia, insulin resistance, and β-cell expansion were more pronounced in BKO mice. In turn, leptin-induced food intake inhibition and hypothalamic insulin signaling were impaired in BKO mice, regardless of the diet, in accordance with deregulation of the expression of hypothalamic neuropeptide genes. Importantly, BKO mice already showed increased β-cell proliferation and glucose-stimulated insulin secretion with respect to WT littermates after two weeks of HFD feeding, before the onset of obesity. ConclusionsCollectively, these results reveal that BACE2 suppression in an obesogenic setting leads to exacerbated body weight gain, hyperinsulinemia, and insulin resistance. Thus, we conclude that inhibition of BACE2 may aggravate the adverse metabolic effects associated with obesity.

Estrogen Regulates the Trafficking of Leptin Receptors

During menopause, when endogenous estrogen levels are depleted, a rise in hypertension is observed in 75% of post-menopausal women, suggesting there is a cardioprotective effect from estrogen. In previous studies, estrogen has been shown to effect hypothalamic paraventricular nuclei by its action to lessen the impact of a glutamate induced increase in blood pressure. These data, along with others, suggest that the cardioprotective effect of estrogen is regionally specific in the central nervous system. However, the mechanism(s) behind estrogen's attenuating effect on blood pressure have not been fully elucidated. We propose that estrogen regulates blood pressure in part, by inhibiting the actions of leptin. Leptin is an adipocyte-derived peptide hormone. Activation of its signaling pathway within the central nervous system can alter blood pressure. Studies with injections of leptin in rodent's dorsomedial hypothalamus showed an increase in blood pressure, while antagonism of leptin receptors correlated with diminished blood pressure. Both results suggest that adjustment of leptin signaling in the dorsomedial hypothalamus is sufficient to alter blood pressure. In this study, we hypothesize that estrogen can block leptin receptor activation and downstream signaling events by increasing the expression of SOCS3 (suppressor of cytokine signaling 3), a negative modulator of leptin signaling. We tested our hypothesis by utilizing an in vitro system (i.e. GT1-7 mouse hypothalamic neuronal cells) and an in vivo system (i.e. female C57B6 mice) that were treated with estradiol (100nM). Our data demonstrated that administering estrogen decreased the expression of leptin receptors in the hypothalamus and we proposed this occurred due to the increase in SOCS3 expression we observed when estradiol was administered. Taken together, these data suggest that estrogen can attenuate leptin's physiological response on blood pressure by increasing an inhibitor (i.e. SOCS3) of leptin's signaling.

Rapid responses of adipocytes to iron overload increase serum TG level by decreasing adiponectin.

Iron overload is tightly connected with metabolic disorders. Excess iron in the adipose and its roles in dyslipidemia are of interest to be identified. In acute iron overload mice receiving intraperitoneal injection of 100 mg/kg/day dextran-iron for 5 days, the epididymis adipose showed a remarkable increase in iron. Serum triglyceride and low-density lipoprotein cholesterol (LDL-C) levels were increased and high-density lipoprotein cholesterol (HDL-C) level was decreased, while serum alkaline phosphatase, aspartate aminotransferase, glucose, and insulin were not affected. The serum-cytokine-microarray showed that adipocytokines, including adiponectin, leptin, and resistin were significantly decreased. Other serum cytokines, including pro-insulin cytokines, inflammatory cytokines, chemokines, and growth factors were not changed, except that ghrelin and chemokine RANTES were increased. Iron overload decreased expressions of adiponectin and leptin both in vivo and in vitro. Intraperitoneal injection of recombinant leptin at 1 μg/g in acute iron overload mice had no significant effects on serum levels of TC, TG, HDL-C, and LDL-C, while intraperitoneal injection of recombinant adiponectin at 3 μg/g partially restored serum TG level through improving activities of lipoprotein lipase and hepatic lipase, but abnormal serum LDL-C and HDL-C were not redressed, suggesting other mechanisms also existed. In conclusion, the adipose responds to iron overload at an early stage to interfere with lipid metabolism by secreting adipocytokines, which may further affect glucose metabolism, inflammation, and other iron overload-induced effects on the body.

Peripheral leptin signaling persists in innate immune cells during diet-induced obesity.

Leptin is a pleiotropic adipokine that regulates immunometabolism centrally and peripherally. Obese individuals present increased levels of leptin in the blood and develop hypothalamic resistance to this adipokine. Here we investigated whether leptin effects on the periphery are maintained despite the hypothalamic resistance. We previously reported that leptin injection induces in vivo neutrophil migration and peritoneal macrophage activation in lean mice through TNF-α- and CXCL1-dependent mechanisms. However, leptin effects on leukocyte biology during obesity remain unclear. In this study, we investigated the in vivo responsiveness of leukocytes to i.p. injected leptin in mice with diet-induced obesity (DIO). After 14-16wk, high-sucrose, high-fat diet (HFD)-fed mice showed hyperglycemia, hyperleptinemia, and dyslipidemia compared to normal-sucrose, normal-fat diet (ND). Exogenous leptin did not reduce food intake in DIO mice in contrast to control mice, indicating that DIO mice were centrally resistant to leptin. Regardless of the diet, we found increased levels of TNF-α and CXCL1 in the animals injected with leptin, alongside a pronounced neutrophil migration to the peritoneal cavity and enhanced biogenesis of lipid droplets in peritoneal macrophages. Supporting our in vivo results, data from ex vivo leptin stimulation experiments confirmed hypothalamic resistance in DIO mice, whereas bone marrow cells responded to leptin stimulation through mTOR signaling despite obesity. Altogether, our results show that leukocytes responded equally to leptin in ND- or HFD-fed mice. These results support a role for leptin in the innate immune response also in obesity, contributing to the inflammatory status that leads to the development of metabolic disease.