Hexokinase

A blood sample was collected after an overnight fast of >8 h. Plasma glucose levels were measured using a hexokinase enzymatic reference method. Fasting insulin levels were measured using a radioimmunoassay (RIA) method (Coat A Count Insulin, Los Angeles, USA). Fasting lipids were analyzed, and for the present study serum levels of cholesterol ≥5.172 mmol l^-1 and triglycerides ≥1.7 mmol l^-1 were considered abnormal.
HOMA-IR was used to evaluate insulin resistance (fasting serum insulin (μU/ml) × fasting plasma glucose (mmol l^-1)/22.5) [27 (link)].

Following the standardized study protocol, data were collected via detailed questionnaires, physical examination, and blood samples during baseline and follow-up visits at a local clinic. Trained clinical staff administered standardized questionnaires and conducted in-person interviews to collect information on demographic data and medical, family, and medication history. Blood pressure, height, body weight, and waist circumference were measured according to standard procedures, and body mass index (BMI) was calculated as weight (kg)/height squared (m²).
At each clinic visit, a 75-g oral glucose tolerance test (OGTT) was performed after collecting a fasting blood sample. FPG, HbA1c, fasting lipids, and 2hPG levels were also measured. After collecting venous blood, all samples were immediately placed on ice to maintain stability. Thereafter, the samples were instantly transported to the laboratory at the First Medical Centre of Chinese PLA General Hospital and processed within 2 h of blood collection. For plasma glucose (including FPG and 2hPG), blood samples were collected in tubes containing sodium fluoride and measured using the hexokinase method. HbA1c was measured using high-performance liquid chromatography (VARIANT II system, Bio-Rad, Hercules, CA). Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were determined using an auto-analyzer (ARCHITECT c16000 System; Abbott Laboratories, Chicago, IL). The quality control protocol for laboratory assays has been published in detail elsewhere (17 (link)).
The diagnosis of dysglycemia (including pre-diabetes or diabetes) was based on OGTT, conforming to the American Diabetes Association criteria (1 (link)). Pre-diabetes was defined as follows: FPG: 100–125 mg/dL (5.6–6.9 mmol/L); or 2hPG during 75 g OGTT: 140–199 mg/dL (7.8–11.0 mmol/L). Diabetes was defined as: documented diagnosis of diabetes in medical records or taking glucose-lowering medications; FPG ≥126 mg/dL (7.0 mmol/L); or 2hPG ≥200 mg/dL (11.1 mmol/L) during 75g OGTT. Normoglycemia was described as FPG <100 mg/dl (5.6 mmol/L) with 2hPG <140 mg/dl (7.8 mmol/L) during 75g OGTT. The primary study outcome was the occurrence of diabetes, defined as diagnosed (i.e., physician-diagnosed diabetes or use of antidiabetic medication during follow-up) or undiagnosed (based on the above diabetes criteria).

Blood metabolites were analyzed from a subset of heifers from each respective treatment (N = 30). Blood samples were collected at pasture turnout and at the final day of monitoring via jugular venipuncture into serum tubes (10 mL; Becton Dickinson Co., Franklin Lakes, NJ), allowed to clot for 30 min and centrifuged at 1,500 × g at 4°C for 20 min. Serum was separated and stored in plastic vials at −20°C until further analysis. Serum samples were analyzed for glucose and NEFA. Samples were analyzed using the Synergy H1 Microplate Reader (Biotek, Winooski, VT) with the Infinity Glucose Hexokinase Kit (Thermo Scientific, Waltham, MA) and NEFA-C Kit (WAKO Chemicals, Inc., Richmond, VA). The intra- and interassay CV was 2.62% and 3.41%, for serum glucose, respectively and 7.75% and 8.29%, for serum NEFA, respectively.

Glucose and insulin were measured in plasma on the Cobas 6000 instrument (Roche Diagnostics, USA) using an enzymatic hexokinase assay or electrochemiluminescence, respectively. HbA1c was determined using the HPLC D10 instrument (Bio-Rad, USA). High sensitivity C-reactive protein (hsCRP) was measured in plasma with the immunoturbidometric method assay (Abbott Architect, USA). Fructosamine was measured in serum via colorimetric rate reaction (Roche Diagnostics, USA). Cholesterol, triglyceride and HDL cholesterol concentrations were measured by enzymatic assays (Abbott Architect, USA). LDL was calculated with the following equation:
LDL = (1.06*Chol) – (1.03*HDLC) – (0.117*Trig) – (0.00047*(TRIG*(Chol-HDLC))) + (0.000062*(Trig*Trig)) – 9.44