Andrew J. Ball

Telomere‐independent cellular senescence in human fetal cardiomyocytes

Fetal cardiomyocytes have been proposed as a potential source of cell‐based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT‐PCR. Cells were found to senesce after 20–25 population doublings, as determined by growth arrest, morphological changes and senescence‐associated β‐galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT‐positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase‐positive or ‐negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi‐1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix–loop–helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell‐based therapy for cardiac injury using primary fetal heart tissue.

Desensitization of sulphonylurea- and nutrient-induced insulin secretion following prolonged treatment with glibenclamide

Functional effects of prolonged exposure to the sulphonylurea glibenclamide were examined in a popular clonal pancreatic beta-cell line, denoted as BRIN-BD11. In acute 20-min incubations, 200 microM of tolbutamide or glibenclamide stimulated insulin release from non-depolarized and depolarized cells, which was dramatically reduced following 18-h culture with 100 microM glibenclamide. Sulphonylurea desensitization in non-depolarized cells was reversed following 6-36-h subsequent culture in the absence of glibenclamide. However, desensitization of insulinotropic effects of sulphonylureas in depolarized cells following glibenclamide culture and associated decline in cellular insulin content was not fully reversible. Culture with 100 microM glibenclamide also markedly reduced the acute insulinotropic actions of glucose, L-alanine, L-arginine, 2-ketoisocaproic acid (KIC) and KCl. These effects were almost completely reversed following 18-h culture in the absence of the sulphonylurea.

Induced desensitization of the insulinotropic effects of antidiabetic drugs, BTS 67 582 and tolbutamide.

Acute and chronic mechanisms of action of novel insulinotropic antidiabetic drug, BTS 67 582 (1,1‐dimethyl‐2‐(2‐morpholinophenyl)guanidine fumarate), were examined in the stable cultured BRIN‐BD11 cell line. BTS 67 582 (100–400 μM) stimulated a concentration‐dependent increase (P<0.01) in insulin release at both non‐stimulatory (1.1 mM) and stimulatory (8.4 mM) glucose. Long‐term exposure (3–18 h) to 100 μM BTS 67 582 in culture time‐dependently decreased subsequent responsiveness to acute challenge with 200 μM BTS 67 582 or 200 μM tolbutamide at 12–18 h (P<0.001). Similarly 3–18 h culture with the sulphonylurea, tolbutamide (100 μM), also effectively suppressed subsequent insulinotropic responses to both BTS 67 582 and tolbutamide. Culture with 100 μM BTS 67 582 or 100 μM tolbutamide did not affect basal insulin secretion, cellular insulin content, or cell viability and exerted no influence on the secretory responsiveness to 200 μM of the imidazoline, efaroxan. While 18 h BTS 67 582 culture did not affect the insulin‐releasing actions (P<0.001) of 16.7 mM glucose, 10 mM arginine, 30 mM KCl, 25 μM forskolin or 10 nM phorbol‐12‐myristate 13‐acetate (PMA), significant inhibition (P<0.001) of the insulinotropic effects of 10 mM 2‐ketoisocaproic acid (KIC) and 10 mM alanine were observed. These data suggest that BTS 67 582 shares a common signalling pathway to sulphonylurea but not imidazoline drugs. Desensitization of drug action may provide an important approach to dissect sites of action of novel and established insulinotropic antidiabetic agents.

Acute and long-term effects of nateglinide on insulin secretory pathways

Acute and chronic effects of the insulinotropic drug nateglinide upon insulin release were examined in the BRIN‐BD11 cell line. Nateglinide (10–400 μM) stimulated a concentration‐dependent increase (P<0.05–P<0.001) in insulin release at a non‐stimulatory (1.1 mM) glucose concentration. The insulinotropic response to 200 μM nateglinide was increased at 30 mM (P<0.01), but not 5.6–16.7 mM glucose concentrations. In depolarized cells, nateglinide (50–200 μM) evoked KATP channel‐independent insulin secretion (P<0.05–P<0.001) in the absence and presence of 5.6–30.0 mM glucose (P<0.001). Exposure for 18 h to 100 μM nateglinide abolished the acute insulinotropic effects of 200 μM nateglinide, tolbutamide or glibenclamide, but had no effect upon the insulinotropic effect of 200 μM efaroxan. While 18 h exposure to 100 μM nateglinide did not affect basal insulin release or insulin release in the presence of 16.7 mM glucose, 25 μM forskolin or 10 nM PMA, significant inhibition of the insulinotropic effects of 20 mM leucine and 20 mM arginine were observed. These data show that nateglinide stimulates both KATP channel‐dependent and‐independent insulin secretion. The maintained insulinotropic effects of this drug with increasing glucose concentrations support the antihyperglycaemic actions of nateglinide in Type II diabetes. Studies of the long‐term effects of nateglinide indicate that nateglinide shares signalling pathways with sulphonylureas, but not the imidazoline efaroxan. This may be significant when considering a nateglinide treatment regimen, particularly in patients previously treated with sulphonylurea.

Specific desensitization of sulfonylurea- but not imidazoline-induced insulin release after prolonged tolbutamide exposure.

Functional effects of prolonged exposure to the sulfonylurea, tolbutamide, were examined in the clonal electrofusion-derived BRIN-BD11 cell line. In acute 20-min incubations, 50-400 microM tolbutamide stimulated a dose-dependent increase (P < 0.01) in insulin release at both non-stimulatory (1.1 mM) and stimulatory (8.4 mM) glucose. Culture with 100 microM tolbutamide (18 hr) caused a marked (67%) decrease in subsequent insulin-secretory responsiveness to acute challenge with 200 microM tolbutamide, though notably, tolbutamide culture exerted no influence on 200 microM efaroxan-induced insulin secretion. Duration of exposure (3-18 hr) to 100 microM tolbutamide in culture also time-dependently influenced subsequent responsiveness to acute tolbutamide challenge, with progressive 47-58% decreases from 6-18 hr (P < 0.001). Similarly, 6- to 18-hr culture with 100 microM efaroxan specifically desensitized efaroxan-induced insulin release. Tolbutamide- and efaroxan-induced desensitization exhibited a time-dependent reversibility, with a sustained return to full insulin-secretory responsiveness by 12 hr. Notably, 18-hr culture with tolbutamide or efaroxan did not significantly affect insulinotropic responses to 16.7 mM glucose, 10 mM 2-ketoisocaproic acid, 10 mM alanine, 10 mM arginine, or 30 mM KCl. Diverse inhibitory actions of tolbutamide or efaroxan culture on late events in stimulus-secretion coupling reveal that drug desensitization is both a specific and important phenomenon. As such, the model system described could prove an important tool in determining the complex modes of action of established and novel clinically useful insulinotropic compounds.