Lantus

Insulin glargine (Lantus; Sanofi‐Aventis, Shanghai, China) was administered initially at a dose of 0.2 IU/kg. The dose was then adjusted according to Table 1. The OADs were supplemented if the glycemic target was not achieved or maintained. The OADs were prescribed in accordance with the algorithm used for the OAD group.

Written informed consent was obtained from owners of all enrolled cats. Cats had a diagnosis of acromegaly on the basis of appropriate clinical history, serum IGF1 concentration >1000 ng/mL (reference interval, 200 to 700 ng/mL), which has a 95% positive predictive value for acromegaly [12 (link)], and pituitary enlargement diagnosed using intracranial imaging (contrast enhanced CT) or postmortem examination [12 (link)]. All acromegalic cats had concurrent diabetes mellitus that was likely to be secondary to acromegaly, and they were receiving lente insulin (Caninsulin; MSD Animal Health, Kenilworth, NJ), protamine zinc insulin (ProZinc; Boehringer Ingelheim, Ingelheim am Rhein, Germany), or glargine insulin (Lantus; Sanofi, Paris, France) (HST group). Nonacromegalic cats who did not have a clinical history consistent with acromegaly or pituitary enlargement, but had undergone postmortem examination and whose owners consented to be enrolled in the study, were consecutively recruited. All cats had previously been patients of the Queen Mother Hospital for Animals (RVC), Beaumont Animals’ Hospital (RVC), or People’s Dispensary for Sick Animals in London, United Kingdom. All cats had been neutered, which is common in the United Kingdom for patient health and population control.

MSCs and ACs were cultured as 3D pellets (5 × 10⁵ cells/pellet) in chondrogenic medium consisting of high-glucose DMEM, 0.1 µM dexamethasone, 0.17 mM ascorbic acid-2 phosphate, 4 mM sodium pyruvate, 0.35 mM proline, 5 µg/mL transferrin, 5 ng/mL selenous acid, 1.25 mg/mL bovine serum albumin (all from Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin, 1% ITS+ premix (Corning Life Sciences, New York City, NY, USA) or similar amounts of insulin (Lantus, Sanofi-Aventis, Frankfurt, Germany), and 10 ng/mL recombinant human TGFβ1 (Biomol, Hamburg, Germany) for up to 6 weeks with medium changes three times a week.
For the indicated time points during MSC chondrogenesis, the chondrogenic medium was supplemented with the BMP inhibitor LDN-212854 (LDN-21; 500 nM in DMSO solvent, day 0–42; Sigma-Aldrich, St. Louis, MO, USA), the WNT-inhibitor IWP-2 (2 μM in DMSO solvent, day 14–42), or LY294002 (LY; 0.25 µM–25 µM in DMSO, day 21–42; both from Tocris Bioscience, Bristol, United Kingdom). When appropriate, the controls were treated with DMSO. Where indicated, TGFβ1 was withdrawn from day 21 onward. For the Western blot detection of phosphorylated AKT, pellets were harvested at the designated days 48 h after the last medium exchange.