Insulin glargine

Six-week-old male db/db mice were purchased from CLEA Japan Inc (Tokyo, Japan). Mice were maintained in a pathogen-free facility under controlled environmental conditions and exposed to a 12:12 h light:dark cycle. After 2 weeks of acclimation, mice were fed HF diets (HFD-32; CLEA Japan Inc., Tokyo, Japan) with or without TA-1887 treatment (0.01% w/w in chow). To assess effects of chronic insulin treatment, animals attached to either insulin or normal saline pumps (Alzet, model 2002; DURECT, Cupertino, CA) were similarly fed and received insulin (3 μg/g/day) or control saline, respectively. Blood glucose levels of insulin-treated mice were adjusted to ~200 mg/dl by additional administration of long-acting insulin (Insulin Glargine, Sanofi, Gentilly, France). Animal experiments were approved by the institutional review board at Kumamoto University, and all animals received humane care.

To elucidate the underlying mechanisms of imeglimin’s effect on β cells, 6- and 8-week-old db/db mice were utilized. The 8-week-old db/db mice were treated with 1-week treatment of vehicle, imeglimin, or insulin glargine (Sanofi-Aventis, Paris, France); 6-week-old db/db mice were treated with vehicle for 1 week. In order to clarify the imeglimin’s efficacy on BCM via mitochondrial involvement in glucose-dependent or -independent manner, the insulin glargine was subcutaneously injected once daily with dose adjustments to achieve glycemic control comparable to that of the imeglimin-treated group. The mice were sacrificed, and the islets isolated by collagenase digestion, as previously described (25 (link)), and subsequently subjected to mitochondrial membrane potential (MMP) and cytosolic cytochrome c measurements. In addition, to assess glucose-dependent and –independent mitochondrial involvement of imeglimin action on β cells, the isolated islets from 6- and 8-week-old db/db mice were incubated in different glucose concentrations of RPMI 1640 (10% FBS, 10 mM HEPES, 5 mM NaHCO₃, 1 mM sodium pyruvate, 100 mg/ml streptomycin, 100 U/ml penicillin and 11.1 mM or 33.0 mM glucose) with DMSO or 100 µM imeglimin for 24 hours at 37°C and 5% CO₂ in a humidified incubator, and subjected to MMP measurement with JC-1 dye.

Patients considered to be undergoing SIIT to reach their glycemic target (FPG 4.4–6.0 mmol/L, 2-hour post-prandial plasma glucose [PPG]: 4.4–7.8 mmol/L) were included in the study. Insulin Lispro (Humalog Kwikpen, Eli Lilly and Company, USA) or Insulin Aspart (Novorapid Flexpen, NovoNordisk, Bagsværd, Denmark) were the fast-acting analog insulins used. Insulin Glargine (Lantus Solostar, Sanofi Aventis, France) and Insulin Detemir (Levemir Flexpen Novo Nordisk, Bagsvaerd, Denmark) were the basal long-acting insulins used. Patients who administered a total daily insulin dose of 0.4–0.5 IU/kg, 50% of which was long-acting basal insulin and 50% fast-acting analog insulin were included in the study. The participants were taught to inject their short-acting insulin before each meal and their intermediate-acting insulin at bedtime. The type of insulin used was not included in the analysis.

For 14 days, 35,000 MSC were cultivated in adipogenic induction medium (87.8% DMEM high glucose, 8.8% FCS, 4 ng/mL insulin glargine (Sanofi-Aventis, Frankfurt am Main, Germany) 90 µg/mL penicillin/streptomycin, 2.2 µg/mL amphotericin B, 0.05 mM isobutyl methylxanthine, 1 µM dexamethasone (all Sigma-Aldrich, Steinheim, Germany), followed by Oilred-O staining and counterstaining with haemalaun solution (both Waldeck, Muenster, Germany). To confirm adipogenic differentiation, the presence of red-stained fatty vacuoles was examined microscopically. One sample was analyzed for each donor in the individual setting and one sample in total for the pooled setting. Results are shown representatively (Figure 2).

For chondrogenic differentiation, 500,000 MSCs were centrifuged for 5 min at 800× g into a pellet and then incubated in chondrogenic induction medium (94% DMEM high glucose, 40 µg/mL transferrin, 40 µg/mL sodium selenite, 1 µM dexamethasone, 0.17 mM ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/mL bovine serum albumin (all Sigma-Aldrich, Steinheim, Germany), 100 µg/mL penicillin/streptomycin, 2.2 µg/mL amphotericin B, 0.1375 IE/mL insulin glargine (Sanofi-Aventis, Frankfurt am Main, Germany), and 10 ng/mL transforming growth factor β1 (Abcam, Berlin, Germany) for 42 days. Afterwards the pellets were fixed for 2 h in 4% paraformaldehyde (Merck, Darmstadt, Germany) and then dehydrated for 2 h in 70%, 96% and 100% 2-propanol, followed by a 30 min incubation in 100% acetone (all Carl Roth, Karlsruhe, Germany). The pellets were then transferred into paraffin and processed into sections for histological evaluation by Safranin-O/Fast Green (Waldeck, Muenster, Germany) staining. A qualitative analysis for orange stained proteoglycans and glycosaminoglycans as a marker for the development of cartilage tissue was microscopically conducted. One sample was analyzed for each donor in the individual setting and one sample in total for the pooled setting. Results are shown representatively (Figure 2).