To study the expression and distribution of neuropeptide Y (NPY) and long leptin receptor (OB-Rb) in the gastrointestinal tract of giant panda, samples of three animals were collected from the key laboratory for reproduction and conservation genetics of endangered wildlife of Sichuan province, China conservation and research center for the giant panda. Paraffin sections of giant panda gastrointestinal tissue samples were observed using hematoxylin-eosin staining (HE) and strept actividin-biotin complex immunohistochemical staining (IHC). The results show that the intestinal histology of three pandas was normal and no pathological changes, and there were rich single-cell and multi-cell mucous glands, long intestinal villi and thick muscularis mucosa and muscle layer. Positive cells expressing NPY and OB-Rb were widely detected in the gastrointestinal tract by IHC methods. NPY positive nerve fibers and neuronal cell were widely distributed in submucosal plexus and myenteric plexus, especially in the former. They were arranged beaded or point-like shape. NPY positive cells were observed in the shape of ellipse and polygon and mainly located in the mucous layer and intestinal glands. OB-Rb positive cells were mainly distributed in the mucous layer and the laminae propria, especially the latter. These results confirmed that NPY and OB-Rb are widely distributed in the gut of the giant panda, which provide strong reference for the research between growth and development, digestion and absorption, and immune function.
Animals ; China ; Intestines ; metabolism ; Neuropeptide Y ; genetics ; metabolism ; Receptors, Leptin ; genetics ; metabolism ; Ursidae ; genetics ; metabolism

The prevailing model of osteology is that bones constantly undergo a remodeling process, and that the differentiation and functions of osteoblasts are partially regulated by leptin through different central hypothalamic pathways. The finding that bone remodeling is regulated by leptin suggested possible endocrinal effects of bones on energy metabolism. Recently, a reciprocal relationship between bones and energy metabolism was determined whereby leptin influences osteoblast functions and, in turn, the osteoblast-derived protein osteocalcin influences energy metabolism. The metabolic effects of bones are caused by the release of osteocalcin into the circulation in an uncarboxylated form due to incomplete gamma-carboxylation. In this regard, the Esp gene encoding osteotesticular protein tyrosine phosphatase is particularly interesting because it may regulate gamma-carboxylation of osteocalcin. Novel metabolic roles of osteocalcin have been identified, including increased insulin secretion and sensitivity, increased energy expenditure, fat mass reduction, and mitochondrial proliferation and functional enhancement. To date, only a positive correlation between osteocalcin and energy metabolism in humans has been detected, leaving causal effects unresolved. Further research topics include: identification of the osteocalcin receptor; the nature of osteocalcin regulation in other pathways regulating metabolism; crosstalk between nutrition, osteocalcin, and energy metabolism; and potential applications in the treatment of metabolic diseases.
Bone Remodeling/physiology ; Bone and Bones/*metabolism ; *Energy Metabolism ; Humans ; Leptin/metabolism ; Osteocalcin/genetics/*metabolism

Leptin, a peptide hormone secreted by adipocytes in proportion of the amount of energy stored in fat, plays a central role in regulating human energy homeostasis. In addition, leptin plays a significant permissive role in the physiological regulation of several neuroendocrine axes, including the hypothalamic-pituitary-gonadal, -thyroid, -growth hormone, and -adrenal axes. Decreased levels of leptin, also known as hypoleptinemia, signal to the brain a state of energy deprivation. Hypoleptinemia can be a congenital or acquired condition, and is associated with alterations of the aforementioned axes aimed at promoting survival. More specifically, gonadotropin levels decrease and become less pulsatile under conditions of energy deprivation, and these changes can be at least partially reversed through leptin administration in physiological replacement doses. Similarly, leptin deficiency is associated with thyroid axis abnormalities including abnormal levels of thyrotropin-releasing hormone, and leptin administration may at least partially attenuate this effect. Leptin deficiency results in decreased insulin-like growth factor 1 levels which can be partially ameliorated through leptin administration, and leptin appears to have a much more pronounced effect on the growth of rodents than that of humans. Similarly, adrenal axis function is regulated more tightly by low leptin in rodents than in humans. In addition to congenital leptin deficiency, conditions that may be associated with decreased leptin levels include hypothalamic amenorrhea, anorexia nervosa, and congenital or acquired lipodystrophy syndromes. Accumulating evidence from proof of concept studies suggests that leptin administration, in replacement doses, may ameliorate neuroendocrine abnormalities in individuals who suffer from these conditions.
Amenorrhea/metabolism ; Animals ; Female ; Humans ; Leptin/blood/deficiency/genetics/*metabolism ; Male ; Neurosecretory Systems/*metabolism

Obesity has become a severe public health problem across the world, and seriously affects the health and life quality of human beings. Here we generated lepr and mc4r mutant zebrafish via the CRISPR/Cas9 technique, and performed morphological and functional characterizations of those mutants. We observed that there was no significant phenotypic difference between homozygous mutants and wild-type controls before 2.5 months post-fertilization (mpf). However, the adult leprand mc4rindividuals displayed increased food intake, heavier weight, and higher body fat percentage, the characteristics of obesity phenotypes. Blood glucose test showed that overfeeding induced significantly impaired glucose tolerance in adult leprand mc4rzebrafish. Furthermore, we analyzed 76 energy metabolism-related transcripts in leprand mc4rzebrafish livers by using real-time RT-PCR, and compared the results with the published microarray data of Lepmouse livers, and found that the changes in the expression of insulin/IGF signaling (IIS) pathway genes in leprzebrafish and Lepmouse were positively correlated, suggesting that the IIS pathway maintains functional conservation between zebrafish and mammals during the evolution of the obesity-regulating molecule network.
Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Insulin ; metabolism ; Leptin ; Mutation ; Obesity ; genetics ; Receptor, Melanocortin, Type 4 ; genetics ; Receptors, Leptin ; genetics ; Signal Transduction ; Zebrafish ; Zebrafish Proteins ; genetics

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

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

OBJECTIVETo examine the changes of expressions of orexin A, orexin receptor-1 (OX1R), prepro-orexin (Prepro-OX) mRNA, OX1R mRNA and ob-R of hypothalamus in rats with chronic renal failure (CRF).

METHODSSixty-two male Wister rats weighing 200-250 g were divided into three groups, including group 1 (normal, n = 5), group 2 (sham-operated, n = 25) and group 3 (CRF, n = 32). Hypothalamus orexin A was assayed by radioimmunoassay. Serum leptin was assayed by enzyme linked immunosorbent assay. The expression of Prepro-OX mRNA and OX1R mRNA of hypothalamus were measured by reverse transcription polymerase chain reaction, and expression of orexin A, OX1R and ob-R by immunohistochemistry. Automatic biochemical analyzer was used to measure the serum creatinine.

RESULTSHypothalamus orexin A levels were negatively correlated (r = -0.63, P < 0.001) with serum leptin levels in the rats. The expression of hypothalamus Prepro-OX mRNA in CRF rats was significantly lower than that of sham-operation at week 12 (P < 0.01). Hypothalamus Prepro-OX mRNA levels were negatively correlated (r = -0.81, P < 0.001) with the levels of serum leptin and serum creatinine (r = -0.68, P < 0.05) in the rats at week 12. The expression of hypothalamus OX1R mRNA in CRF rats was lower than that of sham-operation at week 12 (P > 0.05). Specific immunoreactivity for orexin A was present in perikeryon of the hypothalamus neuron. Specific OX1R-like immunoreactivity was observed in some nerve fibres. Specific immunoreactivity for ob-R was present in membranes of the hypothalamus neuron. Hypothalamus neurons of orexin A-like specific immunoreactivity in CRF rats were significantly fewer than those in shamoperated rats at week 8. Hypothalamus neurons of OX1R-like specific immunoreactivity in CRF rats were similar to those in sham-operated rat at week 8. Hypothalamus neurons of ob-R-like specific immunoreactivity in CRF rats were significantly more than those in sham-operated rats at week 8.

CONCLUSIONSThe lower hypothalamus orexin A levels may be induced by high serum leptin level in CRF rats. The lower expression of hypothalamus Prepro-OX mRNA in CRF rats may be one of the main causes inducing lower hypothalamus orexin A. The expression of OX1R in hypothalamus neurons is somewhat reduced and the expression of ob-R in hypothalamus neurons is somewhat raised in CRF rats. These remain to be studied further.

Animals ; Carrier Proteins ; genetics ; metabolism ; Hypothalamus ; metabolism ; Intracellular Signaling Peptides and Proteins ; Kidney Failure, Chronic ; metabolism ; Leptin ; genetics ; metabolism ; Male ; Neuropeptides ; genetics ; metabolism ; Neurotransmitter Agents ; genetics ; metabolism ; Orexin Receptors ; Orexins ; Protein Precursors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, Cell Surface ; genetics ; metabolism ; Receptors, G-Protein-Coupled ; Receptors, Leptin ; Receptors, Neuropeptide ; genetics ; metabolism

Leptin, a cytokine predominantly secreted from fat tissue, plays an important role in regulating organism energy balance. Leptin can stimulate lipolysis, but the mechanism is unclear. In order to study the molecular mechanism of leptin stimulating lipolysis, we systemically studied the mRNA expression of key lipolytic enzymes. Morphological observation, Oil Red O staining and RT-PCR were used to identify pig primary adipocytes; commercial kits were used to measure the glycerol and FFA release; Semiquantitative RT-PCR was used to detect the mRNA expression of key lipolytic enzymes. The results showed that 100 nmol/L leptin up-regulated the mRNA expression of ATGL, TGH-2, HSL, MGL and LPL (P<0.01), but down-regulated the Perilipin mRNA expression (P<0.01). At the same time, leptin promoted the glycerol release in a dose dependent manner (P<0.01), but had no effect on the FFA release (P>0.05). These indicate that leptin may mainly stimulate lipolysis in pig primary adipocytes by up-regulating the expression of ATGL, MGL, LPL and down-regulating the expression of Perilipin. The unchanged FFA release may be resulted from Leptin promoting UCPs mRNA expression and increasing FFA expenditure.
Adipocytes ; cytology ; enzymology ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Leptin ; pharmacology ; Lipase ; genetics ; metabolism ; Lipolysis ; drug effects ; Male ; Monoacylglycerol Lipases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Swine

OBJECTIVETo assess the association between leptin gene promoter methylation and serum leptin concentrations in patients with impaired glucose regulation (IGR) and type 2 diabetes mellitus (T2DM).

METHODSMethylation status of leptin gene promoter was determined with methylation-specific polymerase chain reaction. Serum leptin concentrations were determined using enzyme-linked immunosorbent assay.

RESULTSAmong three groups of individuals with different levels of glucose, the methylation rates of leptin gene in IGR and T2DM groups were 43.6 % and 31.5 %, respectively, which were significantly lower than that of healthy subjects (59.2%; Chi-square=22.499 and 5.109, respectively, P<0.05). A lower methylation rate was also observed in T2DM group compared with IGR group (Chi-square=3.962, P<0.05). Leptin levels in both T2DM and IGR groups were elevated compared with normoglycemic subjects, but only T2DM group was significantly higher (q=6.81, P<0.01). Linear regression analysis indicated that serum leptin concentrations has increased along with declining of DNA methylation rate (r=-0.95, P<0.01).

CONCLUSIONLower levels of leptin gene promoter DNA methylation and serum leptin concentrations are associated with the development of diabetes. Measurement of the methylation status of leptin gene promoter and expression can facilitate early intervention of the disease.

DNA Methylation ; Diabetes Mellitus, Type 2 ; genetics ; metabolism ; Female ; Genetic Predisposition to Disease ; Glucose ; genetics ; metabolism ; Humans ; Leptin ; blood ; genetics ; metabolism ; Male ; Middle Aged ; Promoter Regions, Genetic