Leptin, an adipocyte-derived peptide hormone, has been shown to facilitate breathing. However, the central sites and circuit mechanisms underlying the respiratory effects of leptin remain incompletely understood. The present study aimed to address whether neurons expressing leptin receptor b (LepRb) in the nucleus tractus solitarii (NTS) contribute to respiratory control. Both chemogenetic and optogenetic stimulation of LepRb-expressing NTS (NTS^LepRb) neurons notably activated breathing. Moreover, stimulation of NTS^LepRb neurons projecting to the lateral parabrachial nucleus (LPBN) not only remarkably increased basal ventilation to a level similar to that of the stimulation of all NTS^LepRb neurons, but also activated LPBN neurons projecting to the preBötzinger complex (preBötC). By contrast, ablation of NTS^LepRb neurons projecting to the LPBN notably eliminated the enhanced respiratory effect induced by NTS^LepRb neuron stimulation. In brainstem slices, bath application of leptin rapidly depolarized the membrane potential, increased the spontaneous firing rate, and accelerated the Ca²⁺ transients in most NTS^LepRb neurons. Therefore, leptin potentiates breathing in the NTS most likely via an NTS-LPBN-preBötC circuit.
Leptin/pharmacology* ; Membrane Potentials ; Neurons/metabolism* ; Solitary Nucleus/metabolism*

Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.
Apoptosis ; drug effects ; Escherichia coli ; genetics ; Humans ; Leptin ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

Animals ; Leptin ; pharmacology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Superoxide Dismutase ; metabolism

Animals ; Cholesterol ; blood ; Hyperlipidemias ; blood ; drug therapy ; Leptin ; pharmacology ; therapeutic use ; Male ; Mice ; Mice, Inbred Strains

OBJECTIVETo study the effects of topiramate (TPM) and valproate acid (VPA) on serum insulin and leptin levels in young and adult rats.

METHODSThirty healthy female young rats (21 days old) and thirty healthy female adult rats (2 months old) were randomly administered with TPM (50 mg/kg daily), VPA (200 mg/kg daily) or normal saline (control group) by intragastric administration for 5 weeks. After 5 weeks, serum leptin and insulin levels were detected by radioimmunoassay (RIA).

RESULTSSerum leptin and insulin levels in both the young and adult TPM groups were remarkably lower than those of the corresponding control group (P < 0.05). The adult TPM group had significantly lower serum leptin and insulin levels than the young TPM group (P < 0.05). In contrast, serum leptin and insulin levels in both the young and adult VPA groups were remarkably higher than those of the corresponding control group (P < 0.05). The young TPM group had significantly higher serum leptin and insulin levels than the adult TPM group (P < 0.05).

CONCLUSIONSTPM decreases serum leptin and insulin levels in young and adult rats, especially in adult rats. VPA increases serum levels of both in young and adult rats, especially in young rats.

Age Factors ; Animals ; Anticonvulsants ; pharmacology ; Body Weight ; drug effects ; Female ; Fructose ; analogs & derivatives ; pharmacology ; Insulin ; blood ; Leptin ; blood ; Rats ; Rats, Sprague-Dawley ; Valproic Acid ; pharmacology

OBJECTIVETo evaluate the insulin and leptin resistance of curcumin on simplicity obesity rats.

METHODSAll 50 SPF grade healthy Sprague-Dawley male initial weaning rats were used for two groups in stratified sampling by weight: 30 in treated group and 20 in control group. They were assigned to the following treatment for 8 weeks: the treated group was fed with high-fat food and the control group was fed with normal food. Eight weeks later, adiposity model rats were prepared. Groups: adiposity model rats were divided into 3 groups: model + low curcumin (1.25 g/kg), model + high curcumin (5.00 g/kg) and a model group. In addition, there also had a normal control and a control + high curcumin (5.00 g/kg) group. Ten rats in every group and all given ground feed. After intragastric administration in different doses of curcumin 4 weeks, the effects and pathological changes were observed by the blood sugar, insulin, leptin and TNF-alpha, pathology and transmission electron microscope of pancreatic gland.

RESULTSGiven 4 weeks the different dose of curcumin on the simplicity obesity rats, the significant diminished weight (435.0 +/- 37.6) g and content of lipocyte (4.78 +/- 1.87) g as compared with the obesity model control (492.3 +/- 14.8) g and (8.94 +/- 1.88) g (t values were 4.484 and 4.961 respectively, P < 0.01), level of blood sugar (4.50 +/- 0.09) mmol/L, insulin (7.43 +/- 0.65) mmol/L, leptin (3.40 +/- 0.39) mmol/L and TNF-alpha (2.42 +/- 0.19) ng/ml were significantly decreased than those of adiposity model rats (4.94 +/- 0.12) mmol/L, (9.30 +/- 0.21) mmol/L, (4.40 +/- 0.23) mmol/L and (2.86 +/- 0.49) ng/ml (t values were 8.297, 7.743, 6.247 and 2.368 respectively, P < 0.05), and there was no significant difference with the control group (4.30 +/- 0.14) mmol/L on the level of blood sugar (t = 0.399, P > 0.05). There were a lot of secretory granules with large sphere volume in beta cells of pancreatic island found by transmission electron microscope, and these secretory granules had a higher electron density than those in non-disposed groups.

CONCLUSIONBy diminishing the sediment of fat, relaxing the lymphatic return, and refraining the apoptosis of beta cells, the curcumin might significantly decrease the level of insulin resistance and leptin resistance caused by the high fat diet.

Animals ; Apoptosis ; Curcumin ; pharmacology ; Disease Models, Animal ; Insulin ; metabolism ; pharmacology ; Insulin Resistance ; Islets of Langerhans ; drug effects ; Leptin ; metabolism ; pharmacology ; Male ; Obesity ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells.

METHODSThe in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot.

RESULTSOil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group).

CONCLUSIONSBy downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.

3T3-L1 Cells ; Adipocytes ; drug effects ; metabolism ; Animals ; Cholesterol ; metabolism ; Leptin ; metabolism ; Mice ; PPAR gamma ; genetics ; metabolism ; Sirolimus ; pharmacology

OBJECTIVETo investigate whether or not NADPH oxidase (NOX) participates in leptin-induced reactive oxygen species (ROS) production in hepatic stellate cells (HSC) and to explore the possible mechanism.

METHODSHSC-T6 cells (rat hepatic stellate cells line) were divided into nine groups: Group1: leptin (100 ng/ml) treated; Group2-6: leptin treated together with inhibitors that block different ROS-producing systems: diphenylene-iodonium (DPI) (20 micromol/L), Rotenone (20 micromol/L), Metyrapone (250 micromol/L), Allopurinol (100 micromol/L) and Indomethacin(100 micromol/L); Group7: leptin treated together with Janus kinase (JAK) inhibitor AG490 50 micromol/L; Group8: normal control group (treated DMEM with 0.1% DMSO); Group9: negative control group (untreated). Intracellular ROS levels were measured with dichlorodihydrofluorescein diacetate (DCFH-DA) dye assay by Fluorescence microscope and/or flow cytometry. NOX activity was analyzed by using spectrophotometer to calculate the absorbance of NADPH. The mRNA levels of Rac1 and p22Phox were evaluated by RT-PCR.

RESULTS(1) Leptin increased significantly the ROS production as compared to normal control group (92.91+/-4.19 vs.27.56+/-6.27, P<0.01) in HSC-T6 cells. Both the NADPH oxidase inhibitor DPI and AG490 (50 micromol/L) blocked the ROS production, inhibitors of other ROS producing systems had no significant effect on ROS production induced by lepin (P is more than 0.05). (2) Leptin treated HSC-T6 cells for 1 hour up-regulated the NOX activity significantly compared with that in normal control group [(1.90+/-0.22) pmol.min(-1).mg(-1) vs. (0.76+/-0.06) pmol.min(-1).mg(-1), P<0.05]. Furthermore, the NOX activity increased after being treated with leptin for 12 hours and 24 hours than being treated for 1 hour. Leptin-induced up-regulation of NOX activity was inhibited by pretreatment with DPI or AG490. (3) The RT-PCR results indicated that mRNA expressions of Rac1 and p22Phox in HSC-T6 cells with 12 hours of leptin stimulation increased significantly as compared with normal control group (0.41+/-0.13 vs 0.14+/-0.08, 0.45+/-0.12 vs 0.20+/-0.08, all P<0.05), while the DPI and AG490 had no effect on the mRNA expressions of Rac1 and p22Phox.

CONCLUSIONNOX is the main cellular source of the reactive oxygen species (ROS) generated by HSCs in response to leptin stimulation. The mechanism is probably that leptin can directly activate NOX through JAK signal transduction and hence induce the expression of NOX subunit to promote the activity of NOX which generates considerable ROS in HSC.

Animals ; Cells, Cultured ; Hepatic Stellate Cells ; drug effects ; metabolism ; Leptin ; pharmacology ; NADPH Oxidases ; genetics ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism

Capillaries ; Cells, Cultured ; Down-Regulation ; Endothelial Cells ; metabolism ; Humans ; Leptin ; genetics ; metabolism ; Ophiopogon ; chemistry ; Polysaccharides ; pharmacology ; RNA, Messenger ; genetics ; metabolism

Adipose Tissue ; Animal Feed ; Animals ; Hypothalamus ; drug effects ; physiology ; Leptin ; pharmacology ; Male ; Mice ; Mice, Inbred Strains ; RNA ; analysis