The Tyneside cohort was designed to further study metabolic changes occurring during weight loss and remission of diabetes ( Figure 1 ). Intervention group subjects were classified as responder or non-responder at the end of each phase. Responders were defined as those achieving non-diabetic levels of HbA1c (<6.5%) and blood glucose (<126mg/dl) off any anti-diabetes medication for at least 2 months. The purpose of the Control group was to examine sequential changes over the time course of the study in type 2 diabetic subjects, and participants (n=2) who lost >5kg weight and became non-diabetic were excluded from the analysis. All studies were performed after an overnight fast, and subjects drove or were transported to the MR Centre by taxi to minimise variability of physical activity and stress of travel.

GP practices were randomized to either Intervention or Control groups. Intervention group subjects stopped all anti-diabetic medication on day 1 of the Counterweight Plus weight management programme consisting of 825–853 kcal/d liquid formula diet (Cambridge Weight Plan Ltd., UK) continued for 12-20 weeks, followed by a 2-6 week food reintroduction phase, then ongoing support for weight maintenance. The Control group continued usual diabetes management by their GP practice according to current UK clinical guidelines.

The metabolic study was nested within the cluster-randomised controlled Diabetes Remission Clinical Trial (DiRECT; ISRCTN03267836) (). Ethical approval was obtained from the West of Scotland Ethics Committee, and written informed consent was obtained from all participants. The primary aim of DiRECT was to assess the effect of weight loss by low calorie diet on type 2 diabetes remission in a routine primary care setting. Individuals with type 2 diabetes living in the Tyneside region of England (n=90, 38F/52M, (mean± SD): age 52.8±7.9 years, weight 100.2±16.3kg, BMI 34.7±7.4 kg/m, diabetes duration 3.0±1.7 years, HbA1c 7.5±1.0 %) were recruited by their general practices ( Figure 2 ). Inclusion criteria were diabetes duration of <6 years, age between 20-65 years, HbA1c ≥ 6.5 % (≥6.1% if taking anti-diabetes agents), and BMI of 27-45kg/m. Subjects were not recruited if pregnant, experienced weight loss of more than 5kg within the past 6 months or they have serious health problems. The majority of participants were white European with <2% of other ethnic minorities including Black African and South Asian. Most participants were on glucose lowering medication. Baseline characteristics of the whole DiRECT cohort have been described () and the baseline anthropometric, clinical and metabolic features for geographically defined subgroup who underwent detailed physiologic study are presented in Table 1

Glucose was measured by the oxidase method (Yellow Springs, USA). HbA1c was quantified using HPLC (Tosoh Bioscience, UK). Liver function tests were analysed by standard methods at the Institute of Cardiovascular and Medical Sciences, University of Glasgow. C-peptide, insulin, glucose, NEFA, VLDL1-triglyceride, ketones and other metabolites were analysed at Clinical Pathology Accreditation Laboratory (Newcastle upon Tyne Hospital NHS Foundation Trust, UK) using standard kits as described in the Key Resources Table

A Stepped Insulin Secretion Test with Arginine stimulation (SISTA) was used to quantitate first phase insulin secretion and maximal rate of insulin secretion (). Square wave hyperglycemia (50.4 then 100.8 mg/dl above baseline) was achieved by bolus of 20% Dextrose (Fresenius Kabi Ltd, UK) followed by variable 20% Dextrose infusion for each 30 minute step using an infusion pump (Arcomedical Infusion Ltd, UK). An arginine bolus of 5g L-Arginine hydrochloride 50% (Martindale Pharmaceuticals, UK) was diluted in 10 ml of 0.9% sodium chloride (Fresenius Kabi Ltd, UK), and injected during the second step of hyperglycemia to assess maximal insulin secretory capacity, followed by sampling every 2 min for 10 min. Blood samples for determination of C-peptide concentrations were obtained every 2 min for the first 10 min of each step, then every 5 min. Insulin secretion rates were calculated using a deconvolution method, modelling C-peptide kinetics ().

VLDL1-triglyceride levels were determined from fasting plasma samples taken at each time point. Briefly, the VLDL1-triglyceride production rate was measured by accumulation of plasma VLDL1-triglyceride during competitive blockade of lipoprotein lipase by excess Intralipid (). To do so, 20% Intralipid (Fresenius Kabi Ltd, UK) was injected intravenously as a bolus (0.1 g/kg body mass) followed by continuous infusion of 10% Intralipid at 0.1 g/kg/h by infusion pump (Arcomed Infusion Ltd, UK). Plasma samples were collected at six points over 75 min. After two step centrifugation, to remove blood cells then chylomicrons plus Intralipid particles (Scientific Laboratory Supplies Ltd, UK), the VLDL1 fraction was separated by ultracentrifugation at 278,000g for 98 minutes using the SW 40 Ti swinging-bucket rotor (Beckman Coulter, USA). Triglyceride concentration of this fraction was quantified using the standard method (Roche Diagnostics, UK), and VLDL1-triglyceride production rates were calculated from the gradient of the linear increase in plasma concentration over time.

All participants underwent Magnetic Resonance (MR) quantification of pancreatic and hepatic fat on three occasions: at baseline, following return to isocaloric eating after weight loss and at 12 months ( Figure 1 ). MR data were acquired using a 3T Philips Achieva scanner with six-channel cardiac array (Philips, Netherlands). Data were acquired by three-point Dixon method, with gradient-echo scans acquired during one breath hold (). Hepatic fat content was measured by selecting homogenous regions of interest on five image slices of liver (). Intrapancreatic fat content was quantified using the MR-opsy method optimized to exclude interlobular adipose tissue areas (). Analysis of pancreas fat was carried out by a single observer (AAM) in a blinded manner.

Quantification and Statistical Analysis

Analyses were conducted on all subjects with paired data both before and after weight loss and weight maintenance phases. Data are presented as mean±SEM for normally distributed data and median (range) for skewed data as stated in the Figure legends and main text. Student paired or two-sample t test was used as appropriate for parametric data and Mann Whitney U test for nonparametric data. All statistical analyses including testing the normality of data distribution were performed using Minitab 17 (Minitab, USA) and a P value <0.05 was considered as significant. Paired data were presented in all tables, and the number of subjects in each group is stated in the column headings of each Table. We excluded from the analysis 6 subjects (2 controls who lost weight and became non-diabetic, and 4 intervention participants who changed responder status between 5 and 12 months)

The study was designed to compare change in parameters between responders and non-responders, assuming a 60% rate of return to non-diabetic glucose control and a 25% loss to follow up. It was powered on the most stringent variable (change in pancreas fat) in responders compared with non-responders. The calculated sample size was achieved by randomising a greater proportion of general practices to Intervention in the Tyneside region. As there was 69% remission of diabetes after weight loss and 64% at 12 months, the above assumptions for statistical analysis were satisfied.