Study Design

SISIMAI is a single-arm intervention study to investigate the metabolic action of imeglimin on patients with type 2 diabetes mellitus being conducted within Japan from February 2022 to March 2026. The enrollment period was from February 2022 to March 2024. The ethics committee of Juntendo University approved this study protocol (reference number J21-011). This study was registered in the Japan Registry of Clinical Trials (jRCTs031210600) and is carried out in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants.

Study Population

Twenty-five patients with type 2 diabetes mellitus who periodically attend an outpatient clinic at Juntendo University Hospital, Tokyo, Japan, have been recruited into the study. The hospital provides comprehensive medical care, including specialized outpatient services, emergency care, and inpatient services. The inclusion and exclusion criteria are shown in Table 1. This study includes all eligible patients who provided informed consent.

Table 1 Inclusion and exclusion criteriaObservation Items and Schedule

The observation items and schedules are shown in Fig. 1. Before the start of the study, patient characteristics, including smoking history, cardiac well-being, drug history, and diabetes duration, were collected. All patients will receive imeglimin 1000 mg twice daily for 20 weeks. Patients who were already taking metformin prior to the study will continue their current dosage of metformin throughout the duration of the trial. Clinical outcomes and adherence are ascertained and clinical and biochemical data collected at all visits. The measurements related to the clinical outcomes are as follows.

Fig. 1

Study flow and measurement timeline. X indicates mandatory examinations. □ indicates optional examinations. BMI body mass index, MRI magnetic resonance imaging, MRS magnetic resonance spectroscopy, OGTT oral glucose tolerance test

75-g Oral Glucose Tolerance Test with Double Tracer (U-[13C]glucose Orally and [6,6-2H2]glucose Intravenously)

A 75-g oral glucose tolerance test (OGTT) with double tracers is performed on all patients before, 1 week after, and 20 weeks after the initiation of imeglimin. Briefly, the subjects were instructed to consume a standard weight-maintenance diet during the 3 days immediately before the test and were asked to refrain from alcohol the day before the study. To enhance the accuracy of the study, they were also required to consume the diet provided by the researchers on the day before the test. After overnight fasting, an intravenous cannula is placed in the forearm for tracer infusion. Another catheter is placed in a vein on the contralateral hand and the hand is warmed using a heating device. We then infuse [6,6-2H2]glucose (Cambridge Isotope Laboratories, MA, USA) intravenously as a primed constant infusion (bolus 200× fasting blood glucose [mg/dL]/100 mg/m2 body surface area [BSA]), followed by a constant infusion of 2 mg/m2 BSA/min until the end of the test. To achieve isotope equilibration, we infuse the isotope for 3 h before the baseline determination of plasma glucose enrichment (− 180 to 0 min). After the basal equilibration period, the participants ingest 75 g glucose containing 1% U-[13C]glucose (Cambridge Isotope Laboratories) (time 0). Blood samples are collected at − 180, − 20, − 10, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min to determine tracer enrichment and glucose, insulin, C-peptide, free fatty acid, and glucagon levels. Glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) levels are also measured at − 180, 0, 15, 30, 45, 60, 90, 120, 180, and 240 min, and oxidized albumin (human non-mercaptalbumin) at − 180 min [15].

After 1 and 20 weeks of imeglimin treatment, patients ingest imeglimin 1000 mg orally 30 min prior to the initiation of the 75-g OGTT. Enrichment of the glucose tracer in plasma is measured using high-performance liquid chromatography (HPLC; LTQ-XL-Orbitrap mass spectrometer; Thermo Fisher Scientific, Waltham, MA, USA), as previously described [16]. Glucose kinetics is calculated as previously described [17, 18]. Endogenous glucose production (EGP), rate of oral glucose appearance (Ra oral), and rate of glucose disappearance (Rd) at each point are calculated, as previously described [16, 17].

Two-Step Hyperinsulinemic-Euglycemic Clamp

A two-step hyperinsulinemic-euglycemic glucose clamp is performed with an artificial endocrine pancreas (STZ-55; Nikkiso Co., Ltd., Tokyo, Japan) before and 20 weeks after the administration of imeglimin. Briefly, the subjects were instructed to consume a standard weight-maintenance diet during the 3 days immediately before the clamp test and were asked to refrain from alcohol the day before the study. To enhance the accuracy of the study, they were also required to consume the diet provided by the researchers on the day before the clamp test. Regular exercise was prohibited for 10 days before the clamp test, and the mean daily physical activity level was estimated over 7 days with an accelerometer (Lifecorder; SUZUKEN Co., Ltd., Aichi, Japan). Subsequently, subjects were instructed to maintain their daily physical activity within ± 10% of their mean daily activity level during the last 3 days, with activity being continuously monitored by the accelerometer. Following an overnight fast, after securing an intravenous cannula in the forearm, primed (200 mg/m2 BSA) [6,6-2H2]glucose is administered intravenously, followed by a constant infusion of 2 mg/m2 BSA per min for 3 h (− 180 to 0 min) to measure fasting EGP [19]. This is followed by a primed insulin infusion (80 mU/m2 per min followed by 40 mU/m2 per min; each lasting 5 min) and continuous insulin infusion at 20 mU/m2 per min for 3 h (first step) (0–180 min) [20]. In the second step, after a priming insulin infusion (160 mU/m2 per min followed by 80 mU/m2 per min; each lasting 5 min), we continuously infuse insulin at 40 mU/m2 per min for 3 h (180–360 min) [20]. The infusion of [6,6-2H2]glucose is decreased by 75% of the initial infusion rate during the first step and 85% of the basal rate during the second step of the clamp to maintain constant plasma glucose enrichment [21]. We enclose the patient’s hand with a warming blanket for arterialization of the hand vein. Plasma glucose levels in arterialized blood are maintained at < 95 mg/dl by a variable 20% glucose infusion containing < 2.5% [6,6-2H2]glucose. Blood samples are withdrawn for biochemical analysis at 10-min intervals during the last 30 min before the clamp and steady-state periods of the first and second steps of the clamp. The enrichment of [6,6-2H2]glucose in the plasma is measured by HPLC with an LTQ-XL-Orbitrap mass spectrometer, as described previously [22]. A steady-state equation is used to calculate the rates of EGP and Rd at each step [23]. Rd values are normalized to fat-free mass (FFM). We use EGP suppression during the first step as an index of hepatic insulin sensitivity, Rd during the second step as an index of muscle insulin sensitivity, and FFA suppression during the first step as an index of adipose tissue insulin sensitivity, as described previously [24]. Insulin clearance is calculated using the following equation [25, 26]: insulin clearance = (IIR/[SSSI − (BSI × SSSC/BSC)]), where IIR = insulin infusion rate, SSSI = steady-state serum insulin concentration during the glucose clamp study, BSI = basal serum insulin, SSSC = steady-state serum C-peptide concentration during the glucose clamp study, and BSC = basal serum C-peptide concentration. The feedback inhibition of insulin secretion is calculated as = 1 − (SSSC/BSC) [27]. Similar methodologies for the two-step hyperinsulinemic-euglycemic glucose clamp measurement have been previously presented in another study [26].

1H-Magnetic Resonance Spectroscopy

1H-Magnetic resonance spectroscopy (VISART EX V4.40; Toshiba Corporation, Tokyo, Japan) scans are performed to measure the intramyocellular lipid (IMCL) values of the right tibialis anterior and soleus muscles and the intrahepatic lipid (IHL) values of liver segment 6 [28, 29] before and after 20 weeks of administration of imeglimin. After the measurements, IMCL is quantified on the basis of the methylene signal intensity (S-fat), with the creatine signal (Cre) as the reference. IMCL is calculated as the ratio of S-fat to Cre. IHL is quantified on the basis of S-fat, with H2O as the internal reference. IHL (%) is calculated as the percentage of H2O + S-fat (S-fat × 100/[H2O + S-fat]) [28, 29].

Fat Distribution

Magnetic resonance imaging is performed for the measurement of visceral and subcutaneous fat areas before and after 20 weeks of imeglimin administration. Briefly, T1 weighted trans-axial scans are obtained and intra-abdominal and subcutaneous fat areas at the fourth and fifth lumbar interspaces are measured, as previously described, using a specific software program (AZE Virtual Place; CANON MEDICAL SYSTEMS CORPORATION, Tokyo, Japan) [29]. Similar methodologies for fat distribution measurement have been previously presented in another study [30].

Peak Oxygen Uptake Test

A cycle ergometer is used to measure the estimated oxygen uptake before and after 20 weeks of imeglimin administration (AEROBIKE 75XL; COMBI Corporation, Tokyo, Japan).

Muscle Biopsy

A needle biopsy of the vastus lateralis muscle is performed under local anesthesia before and after 20 weeks of imeglimin administration. Muscle samples are immediately stored at − 80 °C until use.

Blood and Urine Tests

Non-esterified fatty acids (NEFAs) are measured using enzymatic methods (SEKISUI Medical Co., Ltd., Tokyo, Japan). Plasma insulin and C-peptide levels are measured using a chemiluminescent enzyme immunoassay (FUJIREBIO Inc., Tokyo, Japan) and plasma glucagon, GLP-1, and GIP levels are measured using an enzyme-linked immunosorbent assay (Mercodia AB, Uppsala, Sweden). Oxidized albumin levels are measured using HPLC (SEKISUI Medical Co., Ltd., Tokyo, Japan). HbA1c (glycated hemoglobin), red blood cells, white blood cells, hemoglobin, hematocrit, blood platelet count, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase isozyme, blood urea nitrogen, serum creatinine, total cholesterol, high-density lipoprotein cholesterol, triglyceride, low-density lipoprotein cholesterol, plasma glucose, glycated albumin, spot urine, urinary microalbumin and urinary creatinine are measured using standard methods. The estimated glomerular filtration rate (eGFR) is calculated using the following equation: eGFR = 194 × Scr−1.094 × age−0.287 × (0.739 in women), where Scr = serum creatinine, in which eGFR is expressed as mL/min/1.73 m2 and Scr is expressed in mg/dL.

Blood and urine samples are collected after overnight fasting. GLP-1, GIP, and oxidized albumin levels are measured only at visits 4, 5, and 12, while the other items are measured at visits 4, 6, 7, 8, 9, 10, and 12 (Fig. 1).

Physical Examination

At each visit, body weight, height, body mass index, abdominal circumference, and blood pressure are measured. Body composition (skeletal muscle mass, percent body fat, fat mass, and fat-free mass) is measured using the bioimpedance method (InBody; InBody Japan Inc., Tokyo, Japan).