Incretins

The NHIS was initiated in 1963 in Korea according to the National Health Insurance Act, and all Korean citizens were mandated to participate in this program [12 ]. Currently, the Korean NHIS maintains and manages all databases of Korea’s health service utilization. The detailed structure and function of NHIS is described elsewhere [12 ].
In the present study, we used data from the NHIS-NSC 2002–2013, which were released by the Korean NHIS in 2014. The data comprise a nationally representative random sample of 1,025,340 individuals, which accounts for approximately 2.2 % of the entire population in 2002 [12 ]. The data were built by using probabilistic sampling to represent an individual’s total annual medical expenses within each of 1476 strata defined by age, sex, eligibility status (employed or self-employed), and income level (20 quantiles for each eligibility status and medical-aid beneficiary) combinations via proportional allocation from the 46,605,433 Korean residents in 2002 [12 , 13 (link)]. The NHIS-NSC is a semi-dynamically constructed cohort database; the cohort has been followed up to either the time of the participant’s disqualification from receiving health services due to death or emigration or until the end of the study period, whereas samples of newborn infants are included annually [12 , 13 (link)]. The database contains eligibility and demographic information regarding health insurance as well as data on medical aid beneficiaries, medical bill details, medical treatment, disease histories and prescriptions; such data were constructed after converting insurance claim information to the first day of medical treatment.
From this cohort, we selected subjects recorded to have type 2 diabetes between 2002 and 2004. Type 2 diabetes was defined if anti-diabetic drugs were prescribed and the 10th revision of International Statistical Classification of Diseases, International Classification of Diseases (ICD)-10 codes E11 (non-insulin-dependent diabetes mellitus), E12 (malnutrition-related diabetes mellitus), E13 (other specified diabetes mellitus), or E14 (unspecified diabetes mellitus) was assigned as either principal or additional diagnosis. Antidiabetic drugs dispensed in the pharmacy during the study period in Korea consisted of six classes (i.e., sulfonylureas, biguanide, alpha-glucosidase inhibitor, thiazolidinediones, meglitinide and insulin) [14 (link)]. Incretin-based therapies (i.e. glucagon-like peptide -1 receptor agonists and dipeptidyl peptidase-4 inhibitors) were not introduced during the study period.
This diabetic cohort was followed up from the index date until the end of the study period (i.e., December 31, 2013), until the last year of qualification for those who were alive, or until the date of death for those who died. This study was approved by the NHIS inquiry commission. The personal privacy of each participant was protected by de-identification of the national insurance claims data for analysis. This study was also approved by the Institutional Review Board of the Asan Medical Center (IRB-No 2016-0149).

After the 4 weeks of vildagliptin administration with or without infusions of incretin receptor blockers, the systolic blood pressure (SBP) and pulse rate were measured using indirect tail-cuff equipment. Blood samples were collected after a 6-hour fast. Plasma levels of glucose, total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and nonesterified fatty acids (NEFA) were measured by enzymatic methods. Non-HDL cholesterol was calculated by subtracting HDL cholesterol from total cholesterol. HbA1c was measured by the quick test (A1CNow+ ® 20test-kits; Bayer Yakuhin, Osaka, Japan). Plasma levels of active GLP-1, total GLP-1, total GIP, and insulin were determined by an enzyme-linked immunosorbent assay (ELISA Kit, Millipore, MA; Ultra Sensitive “PLUS” Mouse Insulin ELISA Kit, Morinaga, Yokohama, Japan). Only total GIP was measured, as no test kit for measuring active GIP was commercially available. The plasma levels of total GIP in the Pro³-infused animals remained undetermined, as the test kit for total GIP was cross-reacted with Pro³. Oral glucose tolerance tests were performed on nondiabetic Apoe^−/− mice with or without vildagliptin treatment after a 6-h fast. Glucose (1.5 mg/g body weight) was administered orally through a gavage tube, and blood glucose levels were measured by the glucose oxidase method using the Glucose Monitor System (Sanwa Kagaku, Nagoya, Japan).

The IpGTT was conducted instead of the OGTT because of its ease of use, while also being less stressful for animals than the intragastric gavage technique during OGTT. Add to that, it circumvented the incretin response that might amplify GSIS^[90, ^91] and activate TRPM4/5 channels.^[47] IpGTT was first carried out on three groups of 25 mice per group (three independent experiments): WT, WT with Pyr10 (specific TRPC3 inhibitor),^[24] and Trpc3^−/− mice. Pyr10 (Sigma‐Aldrich, St. Louis, MO, USA) was acutely administered (i.p.) 10 min prior to the IpGTT, at a dose of 8 µg kg⁻¹ by analogy to previously described daily doses.^[14, ^92] For TRPC3 activation testing, two additional mouse groups were used: WT (n = 11) and WT acutely administered (i.p.) 10 min before IpGTT with a specific TRPC3 small‐molecule activator GSK1702934A^[44] (kindly provided by Pr. Klaus Groschner, Gottfried‐Schatz‐Research‐Centre‐Biophysics, Medical University of Graz, Austria) at a dose of 35 µg kg⁻¹ (n = 11). Similarly, insulin secretion was assessed following an i.p. injection of arginine (1 g kg⁻¹) in another set of mice: WT (n = 25), WT previously administered with Pyr10 (n = 25), and Trpc3^−/− mice (n = 25) (three independent experiments). For TRPC6 inhibition experiments, SAR7334 (Tocris Bioscience, Bristol, UK) was used at a dose of 5 mg kg⁻¹ in vivo (i.p.)^[28] and two groups of mice (n = 6 each) were studied, WT and WT SAR7334. Plasma insulin was measured according to Crystal Chem's Ultra‐Sensitive Mouse Insulin ELISA Kit (IL, USA).

Routine biochemical parameters (glucose, HbA1C, insulin, C-peptide) were measured by an automated analyzer Cobas 8000 (Roche) on the day of blood collection. Concentrations of incretins and glucagon were measured in sample aliquots stored at − 80 °C for no longer than 6 months.
Glucose levels were determined using the hexokinase method (GLUC3, Roche Diagnostics GmbH, Mannheim, Germany). Levels of HbA1C were measured by ion exchange chromatography using the Arkray Adams HA-8180 V analyzer (Arkray Corporation, Kyoto, Japan). Insulin and C-peptide levels were determined with commercially available kits (Immunotech, Marseille, France) using an immunoradiometric assay with specific antibodies. Based on fasting glucose and insulin levels the homeostasis model assessment of β-cell function (HOMA-β), homeostasis model assessment of insulin resistance (HOMA-IR) indexes [22 (link)], and quantitative insulin sensitivity check index (QUICKI) [23 (link)] were calculated.
Plasma glucagon, GLP-1, and GIP concentrations were measured by commercial multiplex assay (Human Metabolic Hormone Magnetic Bead Panel, HMHEMAG- 34 K, Merck Millipore, USA). Sensitivity was 13.0 pg/mL for glucagon, 1.2 pg/mL for GLP-1 and 0.6 pg/mL for GIP. Intra- and inter-assay variabilities for the kits were < 10% and 15%, respectively.