Edta coated microvette

Blood samples were collected during GTT in EDTA-coated microvettes (Sarstedt, cat. 16.444.100) and processed by centrifuging at 10,000 rpm for 10 min, and the serum was collected for further analysis. Serum insulin (ng/mL) was measured using the ELISA kit (EMD Millipore, cat. no. EZRMI-13K). The manufacturer’s instructions were followed throughout the analysis. The highest amount of insulin the assay could measure was 10 ng/mL. Considering insulin levels increase after glucose load and development of hyperinsulinemia after HF diet, serum samples were diluted accordingly to be used in the assay. For standard curve preparation, 4-parameter logistic curve fit was used using GraphPad Prism software (version 9.3.1 (350)).

Blood samples from the tail vein were collected in EDTA-coated microvettes (Sarstedt) at 0, 10, and 30 minutes during OGTT and after 5-hour fasting (18 (link)). Plasma was separated from whole blood by centrifugation at 4226g for 10 minutes at 4°C. Insulin and proinsulin were analyzed using mouse ultrasensitive insulin ELISA (ALPCO).

A fasted capillary blood sample was taken for the determination of a range of pro- and anti-inflammatory cytokines (IL-6, IL-1β, IL10 & IL-15), serum brain-derived neurotrophic factor (BDNF) as well as plasma insulin and blood glucose concentrations. Three additional samples were collected at 30-, 60- and 120-min post-breakfast for determination of plasma insulin and blood glucose concentrations.
To increase capillary blood flow, participants' hands were warmed via submersion in warm water prior to sample collection. Blood was collected into 300 μl EDTA coated microvettes (Sarstedt Ltd, UK) for separation into plasma. A single whole blood sample was collected using a pre-calibrated glass pipette (Hawksley Ltd, UK) and immediately deproteinised in 250 μl ice-cooled perchloric acid (2.5%). These samples were centrifuged at 1,000 g for 4 min, at 4°C (Eppendorph 5415C, Hamburg, Germany). Additional blood was collected into a 300 μl microvette, coated in clotting activator, for the separation of serum. The sample was allowed to rest for 30 min at room temperature, before centrifugation at 10,000 × g for 5 min. All samples were frozen immediately at −20°C and transferred to −80°C as soon as possible (typically within ~ 5 h).

Blood samples were obtained by puncture of the right cardiac ventricle and stored at 4°C in EDTA-coated microvettes (Sarstedt, Nümbrecht, Germany). Samples were centrifuged for 10 minutes at 4°C and 2000 rpm, plasma and pellet were stored separately at -80°C. Additionally, brain, liver, heart, and small intestine were dissected, snap frozen over dry ice, and stored at -80°C until further use in this and other studies (see Data availability).

After 4 h fast, blood was collected in EDTA-coated microvettes (Sarstedt) from tail vein and plasma was isolated. Insulin levels from brain lysates (neocortex, hippocampus, and hypothalamus) and plasma were measured using ultrasensitive insulin ELISA (ALPCO Diagnostics). Leptin levels were measured using a Mouse Leptin ELISA Kit (Crystal Chem).

Blood samples were collected in EDTA-coated microvettes® (Sarstedt) and centrifuged at 4 °C, 2500 rpm for 30 min. Plasmas were collected and kept at −20 °C, avoiding repetitive freezing and thawing cycles. Plasma levels of IL6 (Merck), TNFα (Abnova), FGF21 (Abcam), and creatine kinase activity (Merck) were quantified following the manufacturer’s instruction.

Complete blood cell counts were taken from fresh whole blood collected in EDTA-coated microvettes (Sarstedt, Numbrecht, Germany) using an automated cell counter (IDEXX Procyte Dx, IDEXX Laboratories, Hoofddorp, Netherland). The extent of hepatocellular injury and whole-body toxicity was recorded by measuring serum alanine aminotransferase (sALT), serum aspartate aminotransferase (sAST), and lactate dehydrogenase (LDH) activity. A kinetic UV method, optimized by the International Federation of Clinical Chemistry and Laboratory Medicine, was used in an Aries chemical analyzer (Werfen Instrumentation Laboratory S.p.A., Milano, Italy). Values were expressed as Units/Liter (U/L). Each analysis was validated by a certified biochemical chemistry and hematology specialist using quality-controlled blood (CQI) at the San Raffaele Mouse Clinic (http://research.hsr.it/en/services/mouse-clinic/hematologic-testing.html, accessed on 21 October 2021).