Study Population
We enrolled 19 normal subjects and 22 newly diagnosed patients with T2DM at the Nanjing First Hospital between June 2017 and October 2019. The normal subjects were healthy and had no known illness. The patients with T2DM were all newly diagnosed through health screening and had mild diabetes that did not require urgent treatment. The inclusion criteria for the two groups were as follows: 1. normal participants: no previous history of diabetes or any other illness; fasting plasma glucose < 6.1 mmol/L and 2-h plasma glucose < 7.8mmol/L after 75-g OGTTs (the diagnostic criteria of World Health Organization 1999); 2. newly diagnosed patients with T2DM (diagnostic criteria of World Health Organization); HbA1c<86 mmol/mol (10.0%). Both groups had BMI within 18.0~28.0 kg/m2 and were aged 18~60 years. Participants with the following were also excluded: 1. any history of hypoglycemic agent intake or were presently taking hypoglycemic agents; 2. any abnormality in liver or kidney function on blood and urine investigations 3. any history of systemic corticosteroid use in 3 months; 4. any recent infections or acute medical events; 5. pregnancy.
The study was reviewed and approved by the Institutional Ethics Committee of Nanjing First Hospital, Nanjing Medical University, and signed informed consent was obtained from all study participants. This trial was registered in ChiCTR (ChiCTR-OOC-17011643, Registered 12 Jun 2017, http://www.chictr.org.cn/showproj.aspx?proj=19708). This study was carried out in accordance with the Helsinki Declaration, and all methods were carried out in accordance with relevant guidelines and regulations.
OGTT with different temperature
All experiments were conducted at a dedicated facility at the hospital with an ambient temperature of 22˚C. After an overnight fast (>10 hours), half the participants were assigned randomly to either a hot or cold OGTT at 8:00 am, and instructed to take the corresponding hot or cold food throughout the day. The participants then all took the alternative temperature OGTT and food the next day.
Hot and cold glucose solutions were prepared as follows: 75 g anhydrous glucose was dissolved in 300 ml water. Cold glucose solution was cooled in a refrigerator (4 ˚C), while the hot glucose solution was heated in a water bath (55 ˚C). The final temperature of glucose solution was measured just before oral administration to ensure the temperature of 6-8 °C for the cold OGTT and about 50 °C for the hot OGTT. Venous blood was sampled at 0, 5, 10, 30, 60, and 120 min during OGTT. The OGTT was repeated at the opposite temperature the next morning. After the hot OGTT, participants were given hot food for lunch and dinner for the rest of the day and instructed to avoid cool food or water. Similarly, after the cold OGTT, the participants took cold food and water. The food on both days was identical in composition and total energy content to avoid differences in ‘chili’ (capsaicin) related and other gustatory differences. The ‘hot’ foods included hot soup, noodles, rice, vegetables, and meat at temperatures of 45-55 ℃. Cold food was at room temperature of 20-24 ˚C. None of the subjects reported any difficulty with these food temperatures. All meals were consumed within 30 minutes.
Clinical and Laboratory assessments
Height, weight, age, and medical history were collected at the first visit. Body mass index (BMI) was calculated as weight divided by the square of height (kg/m2). Plasma blood glucose levels were measured using the glucose oxidase method with an auto-analyzer (Modular E170, Roche, Mannheim, Germany). HbA1c was measured by high-performance liquid chromatography assay (Bio-Rad Laboratories, Inc., CA, USA). Insulin levels were measured by chemiluminescent immunometric assay using the Modular Analytics E170 (Roche® Diagnostics GmbH, Mannheim, Germany, reference range: 2.3~11.6 mU/L). Plasma cortisol was determined with quantitative radioimmunoassay (Beijing North Institute of Biological Technology, CN). Measurements of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) were made in blood collected in precooled specimen tubes with added protease and DPP-IV inhibitors, and by ELISA assays (USCN LIFE, CN; intra-assay precision: CV<10%; inter-assay precision: CV<12%).
Continuous glucose monitoring
All participants had CGM for 3 consecutive days during this study. A CGM (Medtronic Incorporated, Northridge, CA, USA) sensor was implanted in the anterior abdomen one day before the first OGTT. Participants were instructed in the use of the device and were asked to measure capillary blood glucose four times daily for calibration. Interstitial glucose was continuously measured every 5 minutes (min). The CGM sensor was removed 24 hours after the second OGTT. The participants were asked to maintain their activities without strenuous exercise during CGM for the three consecutive days. Patients with capillary blood glucose ≥ 20.0 mmol/L at any time point were removed from the study. The 24‐hours mean glucose (MBG), standard deviation of MBG (SDBG), coefficient of variation (CV), 24‐hours largest amplitude of glycemic excursion (LAGE), and time in range (TIR) were recorded and compared between the two temperatures in the two groups 10,11.
Statistical analysis
All statistical analyses were performed using the SPSS 22.0 software (SPSS Science, Chicago, USA). All variables were tested for normal distribution of the data and were expressed as mean ± SEM, or as median (IQR). Each of the variables: glucose, insulin, GLP-1, GIP, and cortisol were analyzed by ANOVA for repeated measurements to test for statistical significance of the differences between hot and cold OGTT. If there was statistical significance between the two temperatures (p<0.05), this was followed with a Bonferroni test for specific time points. The paired t-test was used in the comparison of CGM data between cool and hot meals in the same subject. Differences between the groups were examined using the Student's unpaired t-test or Mann-Whitney U-test. All comparisons were 2-sided at a 5% significance level. A p value < 0.05 was considered statistically significant.