Reduced levels of messenger ribonucleic acid for calcium channel, glucose transporter-2, and glucokinase are associated with alterations in insulin secretion in fasted rats.

Abstract

Expression of the genes for voltage-dependent calcium channels (VDCCs), glucose transporter-2 (GLUT2), and glucokinase was studied in pancreatic islets obtained from normal rats after periods of fasting and refeeding using a competitive polymerase chain reaction procedure. A 72-h fast induced about a 3-fold decrease in the beta-cell/neuroendocrine type VDCC alpha 1-subunit and GLUT2 messenger RNA (mRNA) levels and about a 2-fold decrease in insulin and glucokinase mRNA levels compared to those in fed and refed rats. No significant differences were found in beta-actin and the cardiac-type VDCC alpha 1-subunit mRNA levels among fed, fasted, anf refed rats. We also studied insulin secretion from the isolated perfused pancreata obtained from these animals. We found an elevated threshold and decreased insulin release in response to a stepwise increase in glucose concentrations in the isolated perfused pancreata obtained from fasted rats. Fasting also resulted in a dramatic decrease in insulin secretory responses during the application of an L-type VDCC agonist, Bay K8644 (1 microM). Furthermore, fasting resulted in a significant decrease in both 45Ca2+ uptake by the isolated islets and insulin release from the islets. A strong positive correlation was observed between glucose-induced 45Ca2+ uptake and insulin output among the animals studied. On the other hand, after a 24-h refeeding, significant increases in the insulin secretory response to glucose and Bay K8644 were found, with a normalization in mRNA levels for these components. It, thus, appears that the alterations in beta-cell sensitivity to glucose that occur with fasting and refeeding are the result of complex metabolic alterations in the islet associated with reductions in expression of at least in part the beta-cell/neuroendocrine type VDCC in addition to two components of the glucose-sensing apparatus, including glucokinase and GLUT2, and the reduction in mRNA for insulin.

Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat.

“…Finally, these findings in the intact rat provided a clue as to why, in earlier experiments with isolated pancreas preparations, GSIS was invariably found to be severely depressed after simple starvation of the donor animals (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Might this have been due, at least in part, to the fact that the cited studies were always conducted in the absence of FFA?…”

“…One theory ascribed the problem to a starvation-induced loss of glucose-mediated cyclic AMP generation within the ␤ cell (4, 7). Another suggested that starvation brings about inhibition of an early step(s) in glycolysis, in particular glucokinase (5,6,8,11,13), or of glucose transport (13). While such derangements might play some role, what must now be factored into the discussion is that the defect in GSIS caused by food deprivation is completely alleviated if FFA are also present.…”

“…Less well appreciated, however, is the remarkable influence that fasting has on the functioning of the pancreatic ␤ cell. This point, which has been recognized for some three decades (1), and has been reexamined sporadically in the intervening years (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13), is well illustrated by the studies of Burch et al (8). These authors fasted normal rats for increasing periods of time, and then measured the ability of their isolated pancreatic islets to secrete insulin when perifused with a solution containing 10 mM glucose.…”

Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells.

“…To confirm no contamination of genomic DNA, samples without reverse transcriptase treatment were prepared. Oligonucleotide primers used in this study are shown in Table I (22). The reactions were conducted in a DNA Thermal Cycler (Perkin-Elmer Corp., Norwalk, CT) in the presence of [ ␣ -32 P]dCTP under the following conditions: for insulin, glucokinase, and glucose transporter 2 (GLUT2), denaturation at 94 Њ C for 1 min, annealing and extension at 65 Њ C for 2 min, and for glucagon, PP, and ␤ -actin, denaturation at 94 Њ C for 30 s, annealing at 60 Њ C for 30 s, and extension at 72 Њ C for 45 s. The number of cycles was 25 except 23 cycles for glucokinase and 20 cycles for ␤ -actin (19).…”

Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A.

“…Cells were fixed with 3% paraformaldehyde in PBS, treated with 0.1% (vol/vol) Triton X-100 in PBS for 5 min, and incubated sequentially with Blocking Ace (Morinaga, Tokyo, Japan), first antibody, and indocarbocyanine-conjugated donkey anti-rabbit IgG or tetramethylrhodamine isothiocyanateconjugated goat anti-mouse IgG. The (25). In this method, a DNA competitor, which was synthesized by deleting 42 bp from the neuroendocrine/p cell-type VDCC al subunit cDNA by site-directed mutagenesis and which had the same primer sequences as the neuroendocrine/,6 cell-type VDCC al subunit, was used as an internal standard.…”