Human leptin

We collected information by questionnaire, including age, sex, alcohol consumption (drinking alcohol more than 2 days per week or not), and current smoking habits (current smoker or not). Alcohol consumption and current smoking habits were both self-reported. We also recorded the respondents' self-reported sitting time as sedentary time through the questionnaire. Information in medical documents such as DM, HTN, and dyslipidemia was also collected. Body mass index (BMI) was calculated using the formula: weight in kilograms divided by the square of height in meters. Diastolic blood pressure (DBP, mmHg) and systolic blood pressure (SBP, mmHg) were measured more than twice after resting. Waist circumference was defined as the measurement midway between the iliac crest and the last rib, in a horizontal plane, while standing. Participants' biochemical laboratory data were analyzed in the Roche model laboratory at Taiwan E&Q Clinical Laboratory using Roche cobas^® connection modules (CCM). Laboratory data included triglyceride level (TG, mg/dL), fasting plasma glucose (FPG, mg/dL), low-density lipoprotein (LDL-C, mg/dL), high-density lipoprotein (HDL-C, mg/dL), creatinine (mg/dL), and alanine transaminase (ALT, mg/dL). Leptin level (ng/mL) was analyzed by Enzyme-linked Immunosorbent Assay using Invitrogen™ Human Leptin, and the detection range was 0.024–100,000 ng/mL.

The content of the questionnaire included sex, age, drinking status, smoking status, body weight, and body height. The health survey collected hypertension (HTN), diabetes mellitus (DM), and dyslipidemia data. Resting systolic blood pressure (SBP, mmHg) and diastolic blood pressure (DBP, mmHg) were measured at rest at least two times. The following biochemical laboratory parameters were analyzed at the Roche^® model lab at Taiwan E&Q Clinical Laboratory: leptin (ng/mL), estimated glomerular filtration rate (eGFR, ml/min/1.73 m2), creatinine (mg/dl), uric acid (mg/dl), fasting plasma glucose (FPG, mg/dl), and triglycerides (mg/dl). The leptin level (ng/mL) was analyzed by Enzyme-linked Immunosorbent Assay using Invitrogen™ Human Leptin. Body mass index (BMI) was calculated as the person’s weight in kilograms divided by the square of their height in meters.

Serum adiponectin and leptin levels were analyzed using commercially available ELISA kits (Human Adiponectin (Abcam, Cambridge, UK) and Human Leptin (Thermo Fisher Scientific, Waltham, MA, USA)), according to the manufacturer’s instructions. Samples were diluted in 1:2000 for adiponectin and 1:100 for leptin analysis. All serum samples were tested in duplicates and run together with standards on each plate. The plates were placed in the Molecular Device Spectramax M5 microplate reader and the concentration was determined using standard curves built with Softmax Pro 7.1 software. The inter-assay and intra-assay variation were 10% and 15%, respectively.

The activation of mononuclear (MN) cells was performed by incubation with Zymosan in the presence and absence of the exogenous adipokines human adiponectin (Sigma, St Louis, MO, USA) and human leptin (Thermo Fisher, Carlsbad, CA, USA), each at a concentration of 100 ng/mL. The concentrations were in accordance with data from the scientific literature [25 (link)], and preliminary pilot tests were conducted to standardize the concentrations used.
The MN cells were incubated with Zymosan (for 2 h at 37 °C under gentle shaking) and treated with 199 medium (negative control), adiponectin, leptin, and adiponectin+leptin.
The phagocytosis assays were performed with Zymosan pHrodo Green™ (Thermo Fisher, Carlsbad, CA, USA), because it emits green fluorescence in the presence of an acidic pH during the phagocytosis process. Free radical release, apoptosis, and intracellular calcium assays were performed with Zymosan (Sigma, St Louis, MO, USA) without conjugated fluorochrome to avoid interference in the fluorescence intensity of the reagents used in each assay.