The transcription factors Stat5a/b are not required for islet development but modulate pancreatic beta-cell physiology upon aging

Abstract

In insulinoma cell lines proliferation and insulin gene transcription are stimulated by growth hormone and prolactin, which convey their signals through the transcription factors Stat5a and 5b (referred to as Stat5). However, the contribution of Stat5 to the physiology of beta-cells in vivo could not be assessed directly since Stat5-null mice die perinataly. To explore the physiological role of Stat5 in the mouse, the corresponding gene locus targeted with loxP sites was inactivated in beta-cells using two lines of Cre recombinase expressing transgenic mice. While the RIP-Cre transgene is active in pancreatic beta-cells and the hypothalamus, the Pdx1-Cre transgene is active in precursor cells of the endocrine and exocrine pancreas. Mice carrying two floxed Stat5alleles and a RIP-Cre transgene developed mild obesity, were hyperglycemic and exhibited impaired glucose tolerance. Since RIP-Cre transgenic mice by themselves display some glucose intolerance, the significance of these data is unclear. In contrast, mice, in which the Stat5 locus had been deleted with the Pdx1-Cre transgene, developed functional islets and were glucose tolerant. Mild glucose intolerance occurred with age. We conclude that Stat5 is not essential for islet development but may modulate beta-cell function.

Figure 1

Targeted disruption of the Stat5 genes and assessment of Stat5 deletion in β-cells of Stat5^{fl/fl;RIP-Cre} mice. (A) Schematic of the construct used to generate Stat5^{fl/fl;RIP-Cre} mice. (B) Fluorescence immunohistochemical analysis of Stat5 (red) and glucagon (green) in control (C: Stat5^fl/fl) and Stat5^{fl/fl;RIP-Cre} mice. Pancreata from 7 month old mice were used for immunohistochemical analyses. Residual Stat5b-positive cells in Stat5^{fl/fl;RIP-Cre} mice are non-β-cells. D) Impaired glucose homeostasis in 4–5 month old Stat5^{fl/fl;RIP-Cre} mice and RIP-Cre transgenic mice. Results are expressed as average blood glucose level ± SEM of 6–8 males of each group. E) Insulin release from isolated islets. Islets were isolated from two animals per genotype which were used for glucose tolerance test at 5 months. Insulin secretion was induced by basal (3 mM) and 16.7 mM of glucose. (*, #)P < 0.05; (**, ##)P < 0.01; (***, ###)P < 0.001.

Figure 2

Targeted disruption of the Stat5 genes and assessment of Stat5 deletion in β-cells of Stat5^{fl/fl; Pdx1-Cre} mice. (a) Schematic of the Stat5 locus targeted with loxP sites and deletion of the Stat5 genes using the Pdx1-Cre transgene. (b–c) Assessment of Stat5 protein levels. Pancreata from 3 month old mice were used for immunohistochemical analyses. Sections were stained with antibodies against glucagon (green) and Stat5b (red). Immunofluorescence was viewed under an Olympus BX51 microscope (Olympus America Inc., Melville, N.Y.) and images were captured with a Nikon DXM1200 digital camera (Nikon Inc., Melville, N.Y.). (d–e) Fluorescence immunohistochemical analysis of insulin (green) and glucagon (red) in control (d: Stat5^fl/fl) and Stat5^{fl/fl; Pdx1-Cre} mice (e).

Figure 3

Results from glucose tolerance tests in Stat5^fl/f and Stat5^{fl/fl; PDX1-Cre} mice. Glucose tolerance tests were performed on fasted mice after an i.p. injection of 2 g/kg BW glucose at 10 weeks (a) and 6 months (b) of age. Blood glucose values were measured immediately before and 15, 30, 60, and 120 min after glucose injection. Results are expressed as average blood glucose level ± SEM of 6–8 males of each group. (*) and (**) means P < 0.05 and P < 0.01, respectively, from independent t-tests (unpaired and two-tailed). P-value from ANOVA (analysis of variance between groups, two-way repeated) was 0.8661 at 10 weeks (a) and 0.0007 at 6 months (b) of age. (c–d) Glucose tolerance test was performed on 2–3 month old females before pregnancy (c) and on day 17 of gestation (d). Mice were fasted 9 hours and administrated 2 g/kg BW glucose loads at time zero. All results are reported as mean ± SEM of 5 females of each group. (*) means P < 0.05 from t-tests, but ANOVA shows no significant difference both before pregnancy (P=0.7127) and on day 17 of gestation (P=0.2021).