The interplay of prolactin and the glucocorticoids in the regulation of beta-cell gene expression, fatty acid oxidation, and glucose-stimulated insulin secretion: implications for carbohydrate metabolism in pregnancy

Abstract

Carbohydrate metabolism in pregnancy reflects the balance between counterregulatory hormones, which induce insulin resistance, and lactogenic hormones, which stimulate beta-cell proliferation and insulin production. Here we explored the interactions of prolactin (PRL) and glucocorticoids in the regulation of beta-cell gene expression, fatty acid oxidation, and glucose-stimulated insulin secretion (GSIS). In rat insulinoma cells, rat PRL caused 30-50% (P < 0.001) reductions in Forkhead box O (FoxO)-1, peroxisome proliferator activator receptor (PPAR)-gamma coactivator-1alpha (PGC-1alpha), PPARalpha, and carnitine palmitoyltransferase 1 (CPT-1) mRNAs and increased Glut-2 mRNA and GSIS; conversely, dexamethasone (DEX) up-regulated FoxO1, PGC1alpha, PPARalpha, CPT-1, and uncoupling protein 2 (UCP-2) mRNAs in insulinoma cells and inhibited GSIS. Hydrocortisone had similar effects. The effects of DEX were attenuated by coincubation of cells with PRL. In primary rat islets, PRL reduced FoxO1, PPARalpha, and CPT-1 mRNAs, whereas DEX increased FoxO1, PGC1alpha, and UCP-2 mRNAs. The effects of PRL on gene expression were mimicked by constitutive overexpression of signal transducer and activator of transcription-5b. PRL induced signal transducer and activator of transcription-5 binding to a consensus sequence in the rat FoxO1 promoter, reduced nuclear FoxO1 protein levels, and induced its phosphorylation and cytoplasmic redistribution. DEX increased beta-cell fatty acid oxidation and reduced fatty acid esterification; these effects were attenuated by PRL. Thus, lactogens and glucocorticoids have opposing effects on a number of beta-cell genes including FoxO1, PGC1alpha, PPARalpha, CPT-1, and UCP-2 and differentially regulate beta-cell Glut-2 expression, fatty acid oxidation, and GSIS. These observations suggest new mechanisms by which lactogens may preserve beta-cell mass and function and maternal glucose tolerance despite the doubling of maternal cortisol concentrations in late gestation.

Figure 1

Effects of serum deprivation and reduction in ambient glucose concentrations on β-cell gene expression. INS-1 cells were grown in RPMI 1640 containing 10% FCS and 11.1 mm glucose (growth medium). At 80% confluence the cells were washed and incubated for 24 h in growth medium or in serum-free DMEM (5.5 mm glucose, 0.1% human serum albumin). RNA levels were measured by quantitative PCR. Values for cells incubated in growth medium (serum) were adjusted so that the mean equaled 1.0; values for cells in serum-free medium were calculated as a function of the mean of serum-replete values. The figures show the means ± sem of all data from three independent experiments, each of which contained four flasks per treatment group. Statistically significant differences between the experimental groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2

Effects of serum deprivation on basal and glucose-stimulated insulin secretion. INS-1 832/13 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 in growth medium or basal serum-free medium for 16 h. Insulin secretion was measured as described in Materials and Methods. Values represent the means ± sem of six samples in a representative experiment. The figures show the means ± sem of all data from two independent experiments, each of which contained six wells per treatment group. Statistically significant differences between the experimental groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3

Dose-dependent effects of PRL and glucocorticoids on gene expression in INS-1 cells. INS-1 cells were incubated for 20 h in serum-free basal medium in the presence or absence of rat PRL (10–500 ng/ml, 0.4–20 nm), DEX (0.02–1 μm, 20–1000 nm), hydrocortisone (HC; 15–60 μg/dl, 410–1640 nm), or diluent. mRNA levels were measured by quantitative PCR. 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 figure shows the mean ± sem of four flasks per treatment group. PRL had statistically significant effects (P < 0.01) on gene expression at concentrations exceeding 10 ng/ml (0.4 nm). DEX and HC had statistically significant effects (P < 0.001) on gene expression at all concentrations tested.

Figure 4

Effects of PRL on β-cell gene expression in INS-1 cells and primary rat islets. INS-1 cells (A) or primary rat islets (B) were incubated for 20 h in serum-free DMEM (5.5 mm glucose, 0.1% human serum albumin for INS-1 cells) or serum-free RPMI (6.8 mm glucose, 0.1% human serum albumin for rat islets) in the presence or absence of rat PRL (20 nm). mRNA levels were measured by quantitative PCR. 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 figures show the means ± sem of all data from two (islets) to five (INS-1 cells) independent experiments, each of which contained four flasks per treatment group. Statistically significant differences between the PRL and control (−) groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5

Effects of PRL and DEX on gene expression in INS-1 cells. INS-1 cells were incubated for 20 h in serum-free basal medium (A) or basal medium containing 1% (B) or 10% (C) calf serum in the presence or absence of rat PRL (20 nm), DEX (0.1 μm), a combination of the two, or diluent. mRNA levels were measured by quantitative PCR. 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 figures show the means ± sem of four flasks per treatment group. Statistically significant differences between the experimental (hormone treated) and control groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical differences between the DEX group and the PRL + DEX group are indicated by the P values above the horizontal connecting lines.

Figure 5

Effects of PRL and DEX on gene expression in INS-1 cells. INS-1 cells were incubated for 20 h in serum-free basal medium (A) or basal medium containing 1% (B) or 10% (C) calf serum in the presence or absence of rat PRL (20 nm), DEX (0.1 μm), a combination of the two, or diluent. mRNA levels were measured by quantitative PCR. 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 figures show the means ± sem of four flasks per treatment group. Statistically significant differences between the experimental (hormone treated) and control groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical differences between the DEX group and the PRL + DEX group are indicated by the P values above the horizontal connecting lines.

Figure 6

Effects of PRL and DEX on FoxO1 content, phosphorylation, and cellular distribution. INS-1 832/13 cells were grown in serum-containing medium and then incubated in serum free in the presence of rat PRL (40 nm), DEX (1 μm), a combination of the two, or diluent in basal medium for 15 min or 16 h. Phosphorylated FoxO1 (cyto phos), nuclear FoxO1, and tubulin were detected by Western blot, as described in Materials and Methods. Similar results were obtained in three experiments.

Figure 7

PRL induces STAT5 binding to a consensus sequence on the rat FoxO1 promoter. Nuclear proteins were prepared from INS-1 cells treated with PRL (20 nm) or diluent for 30 min. The proteins were incubated with radiolabeled double stranded oligonucleotides encoding STAT5 consensus sequences in the rat β-casein promoter or the rat FoxO1 promoter (Fig. 6A). Parallel incubations contained a 200-fold excess of cold competitor (CC) double-stranded oligonucleotides encoding the STAT5 sequence in the rat β-casein gene or 2 μg of polyclonal anti-STAT5a antibody (Ab, Fig. 6B). The protein-DNA complexes were separated by PAGE. The shifted bands (arrow) represent binding of STAT5 to DNA. The arrowhead (Fig. 6B) represents the supershifted complex.

Figure 8

Overexpression of STAT5b mimics and potentiates the effects of PRL on β-cell gene expression. INS-1 832/13 cells were transfected with recombinant adenoviruses expressing a constitutively active, murine STAT5b adenovirus, or green fluorescent protein (GFP; control). After 24 h of transduction, the conditioned media was analyzed (A) for STAT5 protein using a polyclonal antibody to STAT5. The cells were washed with basal media and then incubated for an additional 24 h with PRL (20 nm) or diluent. mRNA levels were measured by quantitative PCR. Values in diluent-treated control cells expressing green fluorescent protein (control) were adjusted so that the mean equaled 1.0; values for STAT5b-expressing and hormone-treated cells were calculated as a function of the mean of control values. B, Means ± sem of all data from two independent experiments, each of which contained four flasks per group. Statistically significant differences between the experimental (hormone treated) and control groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical differences between the DEX group and the PRL + DEX group are indicated by the P values above the horizontal connecting lines.

Figure 9

Effects of PRL and DEX on INS-1 cell fatty acid oxidation and esterification. INS-1 cells were incubated for 24 h in serum-free RPMI (11.1 mm glucose, 0.1% human serum albumin) in the presence or absence of rat PRL (20 nm), DEX (1 μm), a combination of the two, or diluent (control). Rates of ¹⁴C-palmitate oxidation and esterification were measured as described in Materials and Methods. Values, expressed as nanomoles per milligram protein per hour, represent the means ± se of all data from two independent experiments, each of which contained six wells per group. Statistically significant differences between the experimental (hormone treated) and control groups are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 10

Effects of PRL and DEX on glucose-stimulated insulin secretion in INS-1 832/13 cells. INS-1 832/13 cells were grown in RPMI 1640 (11.1 mm glucose) supplemented with 10% FBS; at 80% confluence the cells were washed and incubated for 16 h in basal medium (DMEM with 5.5 mm glucose) containing rat PRL (20 nm), DEX (0.1 μm), a combination of the two, or diluent (control), and insulin secretion was measured as described in Materials and Methods. Values represent the means ± se of all data from two independent experiments, each of which contained five samples per group. Statistically significant differences between the experimental (hormone treated) and control groups are indicated by asterisks. *, P < 0.05; **, P < 0.01.