Regulation of islet beta-cell pyruvate metabolism: interactions of prolactin, glucose, and dexamethasone

Abstract

Prolactin (PRL) induces beta-cell proliferation and glucose-stimulated insulin secretion (GSIS) and counteracts the effects of glucocorticoids on insulin production. The mechanisms by which PRL up-regulates GSIS are unknown. We used rat islets and insulinoma (INS-1) cells to explore the interactions of PRL, glucose, and dexamethasone (DEX) in the regulation of beta-cell pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH), and the pyruvate dehydrogenase kinases (PDKs), which catalyze the phosphorylation and inactivation of PDH. PRL increased GSIS by 37% (P < 0.001) in rat islets. Glucose at supraphysiological concentrations (11 mm) increased PC mRNA in islets; in contrast, PRL suppressed PC mRNA levels in islets and INS-1 cells, whereas DEX was without effect. Neither PRL nor DEX altered PC protein or activity levels. In INS-1 cells, PRL increased PDH activity 1.4- to 2-fold (P < 0.05-0.001) at glucose concentrations ranging from 2.5-11 mm. DEX reduced PDH activity; this effect was reversed by PRL. PDK1, -2, -3, and -4 mRNAs were detected in both islets and insulinoma cells, but the latter expressed trivial amounts of PDK4. PRL reduced PDK2 mRNA and protein levels in rat islets and INS-1 cells and PDK4 mRNA in islets; DEX increased PDK2 mRNA in islets and INS-1 cells; this effect was reversed by PRL. Our findings suggest that PRL induction of GSIS is mediated by increases in beta-cell PDH activity; this is facilitated by suppression of PDKs. PRL counteracts the effects of DEX on PDH and PDK expression, suggesting novel roles for the lactogens in the defense against diabetes.

Figure 1

Effect of PRL on basal insulin secretion and GSIS in isolated rat islets. Isolated rat islets were incubated in RPMI 1640 containing 10% FCS and 6.8 mm glucose (growth medium). After 48 h in growth medium, the islets were washed and incubated in serum-free RPMI (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL (20 nm) for 24 h. Insulin secretion was measured as described in Materials and Methods. The figures show the means ± sem of all data from two independent experiments, each of which contained four samples per treatment group. ***, Statistically significant differences between the experimental groups (P < 0.001).

Figure 2

Effects of hormones and varying glucose concentrations on PC mRNA levels in islets and INS-1 cells. A, Islets were incubated for 24 h in serum-free RPMI 1640 containing varying glucose concentrations (2.5, 5.5, and 11 mm, 0.5% BSA) in the presence or absence of rat PRL. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. The control C_T value was 25 (Table 1). Values represent the means ± sem of four samples in a representative experiment. ***, Statistically significant differences between the experimental groups (P < 0.001). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.1% HSA) in the presence or absence of PRL. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01). Similar results were obtained in four independent experiments.

Figure 3

Effects of PRL and DEX on PC mRNA expression in rat islets and INS-1 cells. A, PC mRNA levels were measured in islets incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two (PRL + DEX). mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media (5.5 mm glucose + 0.1% HSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; ***, P < 0.001). Similar results were obtained in four independent experiments.

Figure 4

Effects of PRL, glucose, and DEX on β-cell PDH activity. A, INS-1 cells were grown in RPMI 1640 (11.1 mm glucose) supplemented with 10% FBS (growth medium); at 80% confluence the cells were washed and incubated for 24 h in serum-free basal medium in the presence or absence of PRL (20 nm) at varying glucose concentrations (2.5, 5.5, and 11 mm). PDH enzyme activity was estimated by measuring β-cell radioactive CO₂ production from [1-¹⁴CO₂] pyruvate. Values represent the means ± sem of six samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in three independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal medium in the presence or absence of rat PRL (20 nm), DEX (100 nm), or a combination of the two. Values represent the means ± sem of six samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in three independent experiments.

Figure 5

Effects of PRL and glucose on PDK2 mRNA levels in islets and INS-1 cells. A, Islets were incubated for 24 h in serum-free RPMI 1640 with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.5% BSA). mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. The control C_T value was 25 (Table 1). Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.1% HSA) in the presence or absence of PRL. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in four independent experiments.

Figure 6

Effects of PRL and DEX on PDK2 expression in rat islets and INS-1 cells. A, PDK2 mRNA levels were measured in islets incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media (5.5 mm glucose + 0.1% HSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (***, P < 0.001). Similar results were obtained in four independent experiments. C, Effects of PRL and DEX on PDK2 protein levels in rat islets. Rat islets were incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL (20 nm), DEX (100 nm), or a combination of the two. Protein expression was analyzed by Western blot. The values were corrected for γ-tubulin. Similar results were obtained in three independent experiments.

Figure 7

Effects of PRL and glucose on PDK4 mRNA levels in rat islets. A, Islets were incubated for 24 h in serum-free RPMI 1640 with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.5% BSA). mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. The control C_T value was 21 (Table 1). Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; ***, P < 0.001). Similar results were obtained in four independent experiments. B, PDK4 mRNA levels were measured in islets incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; ***, P < 0.001). Similar results were obtained in four independent experiments.