Insulin content was measured as described before [22 (link)]. Briefly, INS-1E cells were seeded on cell culture 24-well plates at a density of 5 × 105 cells per well and cultured overnight. The cells were washed twice with PBS and incubated further with RPMI-1640 medium containing different concentrations of either TNF-α (0, 2, and 20 ng/ml; R&D Systems, Oxon, UK), or leptin (0, 0.5, and 10 nM; R&D Systems, Oxon, UK), or combination of TNF-α and leptin. After 48 h of incubation, the cells were washed twice with Krebs-Ringer bicarbonate buffer (KRBB, 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, 0.1% BSA, and 10 mM HEPES [pH 7.4]) and starved for 2 hours in KRBB, and then incubated for 1 h in KRB buffer containing 3.3 mM or 16.7 mM glucose. The supernatants were collected and centrifuged (1000 rpm, 5 min) to measure insulin content using rat insulin ELISA kits (Linco Research, Inc., St. Charles, MO, USA) according to the kit's instructions.
Leptin
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Japan, Sweden, Germany
Leptin is a protein hormone produced primarily by adipocytes (fat cells) in the body. It plays a key role in regulating energy homeostasis, appetite, and body weight. Leptin acts on specific receptors in the hypothalamus of the brain to signal the body's energy status and modulate food intake and energy expenditure.
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Lab products found in correlation
Adiponectin, by R&D Systems (38 mentions)
Interleukin 6 (il 6), by R&D Systems (16 mentions)
Tnf α, by R&D Systems (12 mentions)
Igf 1, by R&D Systems (11 mentions)
Insulin, by Mercodia (9 mentions)
Insulin, by Merck Group (7 mentions)
Insulin, by ALPCO (6 mentions)
Cholesterol, by Thermo Fisher Scientific (6 mentions)
Insulin, by Crystal Chem (5 mentions)
Fgf21, by Merck Group (4 mentions)
C reactive protein (crp), by R&D Systems (4 mentions)
Fgf21, by R&D Systems (4 mentions)
Adiponectin, by ALPCO (4 mentions)
Contour glucometer, by Bayer (3 mentions)
Chemerin, by R&D Systems (3 mentions)
Accu chek, by Roche (3 mentions)
Ghrelin, by R&D Systems (3 mentions)
Insulin, by R&D Systems (3 mentions)
Tgf β1, by R&D Systems (3 mentions)
Fibroblast growth factor 21, by Merck Group (3 mentions)
161 protocols using leptin
1
Insulin Secretion in Pancreatic Cells
2
Leptin Signaling Pathway Activation
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Cells were seeded at 2 × 105 in 6 well cell culture plates and grown to 70%-80% confluence. The cells were treated with leptin (1.2 nmol/L) (R and D Systems), or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L) for 24-48 h. Basal cells served as untreated controls. The cells were lysed with RIPA buffer (Sigma) containing protease/phosphatase inhibitors (Thermo Fisher). Proteins were pulled down by Immunoprecipitation. Immunoblotting analysis was performed as described[21 (link)]. The membranes were incubated with Ob-R, cyclin D1, pSTAT3, and STAT3 (Santa Cruz Biotechnology) antibodies overnight at 4 °C. GAPDH (Sigma) was used as a protein loading control. Relative protein levels were determined by Image J software (National Institute of Health, NIH).
3
Serum Biomarker Determination Protocol
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Serum concentrations of leptin, insulin, IGF-1, and adiponectin were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols (leptin, R&D Ann Arbor, MI, USA; insulin, Linco-Millipore Corp., MA, USA; IGF-1, R&D Ann Arbor, MI, USA; and adiponectin, Biovendor, Brno, Czech Republic).
4
Biomarker Profiling for Metabolic Disorders
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IL-6, hepcidin, adiponectin, leptin, and fibroblast growth factor-21 (FGF-21) were analyzed using the respective ELISA kits: IL-6 (DY206-05; R&D Systems, Minneapolis, MN), hepcidin (DY8307-05; R&D Systems, Minneapolis, MN), adiponectin (DY1065-05; R&D Systems, Minneapolis, MN), leptin (DY398-05; R & D Systems, Minneapolis, MN), and e FGF21 (DF2100; R & D Systems, Minneapolis, MN), according to the manufacturer's instruction.
5
Leptin Treatment Effects on Cholangiocarcinoma and Endothelial Cells
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Cell culture and treatments. The human cholangiocarcinoma cell lines SK-ChA-1 and TFK-1 and the human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Medical Sciences. The SK-ChA-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) and TFK-1 cells were cultured in RPMI-1640 (both from Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% foetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 100 mg/ml streptomycin. HUVECs were cultured in endothelial cell growth medium (PromoCell GmbH). All cell cultures were maintained at 37˚C in a humidified atmosphere with 5% CO 2 .
For leptin treatment, cells in the log phase were treated with leptin (R&D Systems, Inc.) at 0, 50, 100 or 200 ng/ml, for 0, 24 or 48 h. The morphology of cells was imaged under a light microscope (magnification, x400).
For leptin treatment, cells in the log phase were treated with leptin (R&D Systems, Inc.) at 0, 50, 100 or 200 ng/ml, for 0, 24 or 48 h. The morphology of cells was imaged under a light microscope (magnification, x400).
6
Exercise-induced Biomarker Dynamics
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Venous blood was obtained from the participants pre- and postexercise intervention, following 12 h of fasting (09:00 a.m.). Blood was collected in plasma-separating tubes, centrifuged at 3000 rpm for 10 min to obtain the plasma, and subsequently stored at −80 °C until analysis. BDNF (R&D Systems, Minneapolis, MN, USA), NGF (R&D Systems, Minneapolis, MN, USA), adiponectin (R&D System, Minneapolis, MN, USA), leptin (R&D System, Minneapolis, MN, USA) and GLP-1 (Cusabio, Wuhan, China) blood levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s instructions. Absorbance was measured using a microplate spectrophotometer (SpectraMax M2e Microplate Reader; CA, USA).
7
Metabolic Hormones and Glucose Response
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Insulin was measured using a chemiluminescent immunoassay (Invitron IV2-001, Invitron, Monmouth, UK). Cross-reactivity with C-peptide and proinsulin was less than 1.5% and assay sensitivity was 1.5 pmol/L. Active ghrelin (Millipore, Merck, UK), PYY (Millipore), total GLP-1 (Millipore) and leptin (R&D Systems, Abingdon, UK) were all measured using ELISA assays. Assay sensitivity for ghrelin was 15 pg/mL; PYY was 6.5 pg/mL; GLP-1 was 1.5 pmol/L and leptin was 7.8 pg/mL.
In non-obese individuals, blood glucose is expected to peak around 60–90 min after a carbohydrate-containing meal and returns to preprandial levels within 180 min. Glucose homeostasis will be abnormal in children with obesity, and varying degrees of postprandial hyperglycaemia may occur. However, RS is expected to improve PYY, GLP1 and gut hormones in a metabolically advantageous manner, and will improve insulin sensitivity and therefore is expected to improve glucose homeostasis.
In non-obese individuals, blood glucose is expected to peak around 60–90 min after a carbohydrate-containing meal and returns to preprandial levels within 180 min. Glucose homeostasis will be abnormal in children with obesity, and varying degrees of postprandial hyperglycaemia may occur. However, RS is expected to improve PYY, GLP1 and gut hormones in a metabolically advantageous manner, and will improve insulin sensitivity and therefore is expected to improve glucose homeostasis.
8
Serum Leptin and Insulin Quantification
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9
Metabolic Profiling of Mice
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Intraperitoneal glucose (1 g/kg, with AUC calculated from the baseline) tolerance tests were performed at 0930, following an overnight (16 h) fast. Glucose was measured with a Glucometer Contour (Bayer, Mishawaka, IN). Insulin (0.75 unit/kg, i.p.) tolerance tests were performed at 0930, in nonfasted mice, with AUC calculated from 0 mg/dl. Blood was collected at 0930 by tail bleed at 17 weeks of age for measurements of fed glucose and insulin, triglycerides, free fatty acids, cholesterol, Leptin, and adiponectin. Free fatty acids (Fujifilm Waco Diagnostics, Mountain View, CA, reagents # 999-34691, 995-34791, 991-34891, 993-35191), triglycerides (Pointe Scientific Inc., Canton, MI, # T7532-120), and cholesterol (Thermo Scientific, Middletown, VA, # TR13421) were measured using the indicated colorimetric assays. Leptin (R&D Systems, Minneapolis, MN, # MOB00), insulin (Crystal Chem, Downers Grove, IL, # 90010 using mouse insulin standard # 90020), and adiponectin (Alpco, Salem, NH, #47-ADPMS-E01) were measured by ELISA.
10
Proliferation Assay on Isolated SP Cells
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Isolated SP cells were seeded (5 × 103 cells) in 96-well plates in RPMI 1640 without phenol red supplemented with FBS 10%, L-glutamine (2 mM), and gentamicin (50 mg/mL). After 24 h, the medium was replaced and cells were treated with Tx (7.5 and 12.5 µM), doxorubicine (Doxo, 0.1 µM), Fv (0.3 nM), leptin (1000 ng/mL) (R&D systems, Abingdon, UK). Cell proliferation was measured by a resazurin fluorescence test (25 µg/mL) at 24 h post-incubation as described previously. Results were expressed in arbitrary units of fluorescence.
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