Differential and complementary effects of glucose and prolactin on islet DNA synthesis and gene expression

Abstract

The mechanisms by which lactogenic hormones promote β-cell expansion remain poorly understood. Because prolactin (PRL) up-regulates β-cell glucose transporter 2, glucokinase, and pyruvate dehydrogenase activities, we reasoned that glucose availability might mediate or modulate the effects of PRL on β-cell mass. Here, we used male rat islets to show that PRL and glucose have differential but complementary effects on the expression of cell cyclins, cell cycle inhibitors, and various other genes known to regulate β-cell replication, including insulin receptor substrate 2, IGF-II, menin, forkhead box protein M1, tryptophan hydroxylase 1, and the PRL receptor. Differential effects on gene expression are associated with synergistic effects of glucose and PRL on islet DNA synthesis. The effects of PRL on gene expression are mirrored by β-cell overexpression of signal transducer and activator of transcription 5b and are opposed by dexamethasone. An ad-small interfering RNA specific for cyclin D2 attenuates markedly the effects of PRL on islet DNA synthesis. Our studies suggest a new paradigm for the control of β-cell mass and insulin production by hormones and nutrients. PRL up-regulates β-cell glucose uptake and utilization, whereas glucose increases islet PRL receptor expression and potentiates the effects of PRL on cell cycle gene expression and DNA synthesis. These findings suggest novel targets for prevention of neonatal glucose intolerance and gestational diabetes and may provide new insight into the pathogenesis of β-cell hyperplasia in obese subjects with insulin resistance.

Fig. 1.

Effect of PRL on gene expression in isolated rat islets. Islets were incubated for 24 or 48 h in serum-free RPMI (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL (20 nm). In each experiment, the groups contained four to six samples per treatment group. mRNA levels were measured by qRT-PCR. Control (diluent treated) values were adjusted so that the means equaled 1.0. Values for PRL-treated islets are expressed as percentage change from controls at 24 and 48 h. Cyc, Cyclin. Data represent mean ± sem of all values obtained in two to five independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 2.

Effect of PRL, STAT5b, and DEX on β-cell gene expression. A, INS-1 832-13 cells in the control (Con) and PRL groups were transfected with an adenovirus containing GFP. Cells in the Stat5b groups were transfected with an adenovirus containing a constitutively active form of Stat5b. The cells were then 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). Values represent mean + se (n = 4) of levels of mRNA relative to controls. Control values were adjusted so that the mean equaled 1.0. Values in other groups were calculated as a function of control values. *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to controls. Similar results were obtained in two 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). mRNA levels were measured by qRT-PCR. Control (diluent treated) values were adjusted so that the mean equaled 1.0. Other values represent the mean + se (n = 4) percentage change relative to controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Similar results were obtained in two experiments. C, Rat islets were incubated for 24 h in serum-free RPMI containing 5.5 mm glucose in the presence or absence of DEX (100 nm). mRNA levels were measured by qRT-PCR. Cyc, Cyclin. Values are expressed as percentage change from controls. Data represent mean ± sem of five values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

Effects of glucose and PRL on [³H]-thymidine incorporation in isolated rat islets. Islets were incubated for 48 h in serum-free RPMI containing 2.5, 5.5, or 17.8 mm glucose in the presence or absence of rat PRL (20 nm). Methyl-[³H]-thymidine was added during the last 4 h of cell culture. [³H]-thymidine incorporated into DNA was normalized to total cellular protein. Values represent the mean + se of 8–12 samples in each group (in a total of three independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001. CPM, Counts per minute.

Fig. 4.

A, Effects of glucose excess (17.8 mm) on gene expression in rat islets. Isolated rat islets were incubated for 24 or 48 h in serum-free RPMI containing 17.8 mm glucose. Additional islets were incubated in medium containing 5.5 mm glucose. Values represent mean + se (n = 4–6) percentage change of levels of mRNA relative to controls (islets incubated in 5.5 mm glucose). *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to controls. Similar results were obtained in three experiments. B, Effects of glucose deprivation (2.5 mm) on gene expression in rat islets. Isolated rat islets were incubated for 24 or 48 h in serum-free RPMI containing 2.5 mm glucose. Additional islets were incubated in medium containing 5.5 mm glucose. Cyc, Cyclin. Values represent mean + se (n = 4–6) percentage change of levels of mRNA relative to controls (islets incubated in 5.5 mm glucose). *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to controls. Similar results were obtained in two experiments.

Fig. 5.

Effects of PRL and glucose in combination on gene expression in rat islets. Isolated rat islets were incubated for 48 h in serum-free RPMI containing varying concentrations of glucose in the presence or absence of rat PRL (20 nm). Values represent mean + se (n = 8–18 in each group in a total of four independent experiments) levels of mRNA relative to controls (islets incubated in 5.5 mm glucose alone). Cyc, Cyclin. Control values were normalized to a mean of 1.0. *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to controls.

Fig. 6.

Effects of cyclin (Cyc) D2 siRNA on cyclin D2 mRNA and [³H]-thymidine incorporation in rat islets. Rat islets were transfected with adenoviruses encoding a scrambled siRNA or an siRNA encoding cyclin D2. The islets were then incubated for 48 h in serum-free RPMI in the presence or absence or rat PRL (20 nm). [³H]-thymidine was added for the final 4 h of incubation. Cyclin D2 mRNA values represent mean + se of four samples normalized to controls (diluent-treated islets transfected with the scrambled siRNA), which were adjusted so that the mean equaled 1.0. Values for [³H]-thymidine incorporation represent the mean + se of 6–12 samples. *, P < 0.05; ***, P < 0.001 relative to controls. Similar results were obtained in two experiments. CPM, Counts per minute.