FoxO1 Plays an Important Role in Regulating β-Cell Compensation for Insulin Resistance in Male Mice

Abstract

β-Cell compensation is an essential mechanism by which β-cells increase insulin secretion for overcoming insulin resistance to maintain euglycemia in obesity. Failure of β-cells to compensate for insulin resistance contributes to insulin insufficiency and overt diabetes. To understand the mechanism of β-cell compensation, we characterized the role of forkhead box O1 (FoxO1) in β-cell compensation in mice under physiological and pathological conditions. FoxO1 is a key transcription factor that serves as a nutrient sensor for integrating insulin signaling to cell metabolism, growth, and proliferation. We showed that FoxO1 improved β-cell compensation via 3 distinct mechanisms by increasing β-cell mass, enhancing β-cell glucose sensing, and augmenting β-cell antioxidative function. These effects accounted for increased glucose-stimulated insulin secretion and enhanced glucose tolerance in β-cell-specific FoxO1-transgenic mice. When fed a high-fat diet, β-cell-specific FoxO1-transgenic mice were protected from developing fat-induced glucose disorder. This effect was attributable to increased β-cell mass and function. Furthermore, we showed that FoxO1 activity was up-regulated in islets, correlating with the induction of physiological β-cell compensation in high-fat-induced obese C57BL/6J mice. These data characterize FoxO1 as a pivotal factor for orchestrating physiological adaptation of β-cell mass and function to overnutrition and obesity.

Figure 1.

Effect of FoxO1 on glucose metabolism and β-cell function in FoxO1-transgenic mice on regular chow. A, Body weight. B, Blood glucose levels. C, Glucose tolerance test. Mice were fasted for 5 hours and were ip injected with glucose (2 g/kg), followed by determining blood glucose levels at different times. D, Insulin tolerance test. E, Basal and GSIS. Mice were fasted for 16 hours, followed by ip injection of glucose (2 g/kg). Plasma insulin levels were determined before (basal condition) and 15 minutes after glucose injection (induced condition). Data in A–E were obtained from FoxO1-tg and WT littermates (male, n = 10/group) on regular chow at 16 weeks of age. F, Islet FoxO1 protein levels. G, Islet mRNA levels. FoxO1-tg and WT littermates (male, 17 wk old, n = 10/group) were euthanized after a 16-hour fasting for isolating islets, which were subjected to real-time RT-PCR analysis using 18S RNA as control or immunoblot assay using rabbit anti-FoxO1 antibody, using anti-β-actin as control. *, P < .05 and **, P < .001 vs WT control. NS, not significant.

Figure 2.

Effect of FoxO1 on glucose metabolism and β-cell function in FoxO1-transgenic mice on high-fat diet. FoxO1-tg and WT littermates (male, 8 wk old, n = 10) were fed a high-fat diet for 8 weeks. A, Body weight. B, Fat mass determined by MRI. C, Lean mass. D, Blood glucose levels. E, Glucose tolerance test. F, Basal and GSIS. G, Ex vivo GSIS. Aliquots of islets (n = 50) isolated from high-fat-fed FoxO1-tg and WT littermates (16 wk old, n = 3/group) were cultured in RPMI 1640 medium overnight, followed by shifting culture medium from 2.8mM to 20mM glucose concentrations. Insulin levels were determined in culture medium collected at 30 minutes in low- vs high-glucose concentrations and were normalized to total islet proteins. H, Insulin tolerance test. Pancreas tissues of high-fat-fed WT (I) and FoxO1-tg (J) mice (n = 10) were subjected to antiinsulin immunohistochemistry for determining islet size (K) and islet mass (L). Additionally, high-fat-fed WT and FoxO1-tg mice (male, 5 wk old, n = 4/group) were treated with BrdU (ip 100 mg/kg daily) for 4 days. Pancreata of WT (M) and FoxO1-tg (N) mice were subjected to anti-BrdU immunohistochemistry for determining BrdU-positive β-cells out of total β-cells per islet (O). *, P < .05 and **, P < .005 vs WT control. Scale bar, 50 μm.

Figure 3.

Effect of FoxO1 on islet mRNA expression in FoxO1-transgenic mice on high-fat diet. FoxO1-tg and WT littermates (male, 8 wk old, n = 10/group) were killed under fasting conditions after 8 weeks of high-fat feeding. Islets were subjected to real-time qRT-PCR analysis using 18S RNA as control. A, Islet mRNA profiles of genes in cell-cycle G₁/S transition. B, The mouse Ccnd3 promoter. Underlined are nucleotides corresponding to the consensus FoxO1 binding site. C, ChIP assay for FoxO1-Ccnd3 promoter interaction. D, Ccnd3 promoter activity in INS-1 cells, as determined by luciferase reporter assay; n = 3. E, Ccnd3 protein levels. Aliquots of islets (n = 150/mouse) from high-fat-fed FoxO1-tg and WT littermates (n = 3/group) were subjected to anti-Ccnd3 immunoblot analysis, using anti-β-actin as control. The amounts of Ccnd3 relative to β-actin proteins in islets were determined. F, Islet mRNA profiles of genes in β-cell mass and function regulation. *, P < .05 and **, P < .005 vs WT control.

Figure 4.

FoxO1 contributes to physiological β-cell compensation for insulin resistance in obese mice. C57BL/6J mice (male, 6 wk old, n = 7/group) were fed regular chow or high-fat diet for 10 weeks, followed by determining body weight and blood glucose metabolism. A, Body weight. B, Fed blood glucose levels. Fed blood glucose levels were determined in mice under ad libitum condition. C, Fasting blood glucose levels. Fasting blood glucose levels were determined after a 16-hour fasting. D, Immunohistochemistry of pancreas for visualizing Pdx1 and FoxO1 proteins in islets. E, Fasting plasma insulin levels. Mice were fasted for 16 hours for determining fasting plasma insulin levels. F, FoxO1 mRNA levels in islets. G, FoxO1 protein levels in islets. H, Islet mRNA levels. Mice were killed at 17 weeks of age for isolating islets. Handpicked islets (150–200 islets per mouse) were subjected to real-time qRT-PCR analysis using 18S RNA as control or immunoblot assay using β-actin as control for determining FoxO1 and β-cell mRNA levels as well as FoxO1 nuclear vs cytoplasmic protein levels. *, P < .05 and **, P < .001 vs control. NS, not significant. Scale bar, 50 μm.

Figure 5.

FoxO1 and Pdx1 colocalize in mouse islets. Male FoxO1-tg (n = 6, 8 wk old) and age/sex-matched WT littermates (n = 6) fed high-fat diet were killed at 20 weeks of age. The pancreas was sectioned for anti-FoxO1 (A and E) and anti-Pdx1 (B and F) immunohistochemistry. C and G, Merged images for illustrating FoxO1 and Pdx1 colocalization in the nucleus of islet cells. D and H, Nuclei of cells stained by DAPI. Scale bar, 50 μm.

Figure 6.

FOXO1 and PDX1 colocalize in human fetal islets. Frozen sections of human fetal pancreas (21 wk old) were coimmunostained with anti-FOXO1 (A) and anti-PDX1 (B) antibodies. Merged images were shown in C. Nuclei of cells were stained with DAPI (D). E–H, Same sections visualized at high resolution. Islets were outlined by dash lines. Islet cells that were positively stained for both FOXO1 and PDX1 in the nucleus were marked by arrows. Scale bar, 100 μm.

Figure 7.

Effect of FoxO1 on Glut2 and Irs2 expression in β-cells. A, Schematic depiction of the mouse Glut2 promoter. The nucleotides of the FoxO1 consensus site were underlined. B, ChIP assay. C57BL/6J mice (male, 10 wk old) were euthanized after a 16-hour fasting, and the pancreases were subjected to ChIP assay using preimmune IgG (n = 3) and anti-FoxO1 antibody (n = 3). The immunoprecipitates were subjected to PCR analysis for detecting FoxO1-bound Glut2 promoter DNA using specific primers flanking the proximal region (−638/−318 nt) of the mouse Glut2 promoter. As input control, aliquots of pancreas cell lysates before immunoprecipitation were subjected to PCR analysis. As off-target control, the immunoprecipitates were analyzed by PCR assay using a pair of primers flanking the distal region (−5004/−4649 nt) of the mouse Glut2 promoter. In addition, INS-1 cells were transduced with Adv-FoxO1 and Adv-Empty control vector (100 pfu/cell). Each condition was run in triplicate. After a 24-hour incubation, cells were processed for real-time qRT-PCR analysis using 18S RNA as control. C, Glut2 mRNA levels. D, FoxO1 mRNA levels. E, Anti-Glut2 immunoblot. Aliquots of islets (n = 150/mouse) from high-fat-fed FoxO1-tg and WT littermates (n = 3/group, 16 wk old) were subjected to anti-Glut2 immunoblot analysis, using anti-β-actin as control. F, Islet Glut2 protein levels. The amount of Glut2 proteins was determined after normalizing to β-actin protein in islets. G, Irs2 mRNA levels. H, Irs1 mRNA levels. *, P < .05 vs control. NS, not significant.

Figure 8.

Effect of FoxO1 on β-cell antioxidative function. A, Effect of FoxO1 on β-cell antioxidative gene expression. Islets from FoxO1-tg and WT littermates (male, 10 wk old, n = 6/group) were subjected to real-time qRT-PCR analysis for determining Sod1, Cat, Gpx1, and FoxO1 mRNA levels. B, Effect of FoxO1 on antioxidative gene expression in INS-1 cells. INS-1 cells were transduced with Adv-Empty and Adv-FoxO1 vectors (100 pfu/cell). C, Immunoblot of INS-1 cells pretransduced with Adv-Empty and Adv-FoxO1 vectors. D, Effect of FoxO1 on antioxidative protein levels in INS-1 cells. After a 24-hour incubation, cells were subjected to real-time qRT-PCR and immunoblot assays. E, The mouse Sod1 promoter. F, ChIP analysis of FoxO1-Sod1 promoter association. G, The mouse Cat promoter. H, ChIP analysis of FoxO1-Cat promoter interaction. Pancreatic islets were subjected to ChIP assay using anti-FoxO1 or anti-β-galactosidase IgG for determining FoxO1-bound DNA. As off-target control, immunoprecipitated DNA was analyzed by real-time PCR, using primers that are complementary to the mouse Sod1 or Cat coding region. I, Sod1 promoter activity. J, Cat promoter activity. The mouse Sod1 (−880/+1 nt) or Cat (−755/+1 nt) promoter was cloned into the pGL3-basic luciferase reporter system for determining Sod1 or Cat promoter activity in INS-1 cells that were pretransduced with Adv-FoxO1 or Adv-Empty vector (100 pfu/cell). K, Effect of FoxO1 on apoptotic gene expression in INS-1 cells. INS-1 cells pretransduced with Adv-FoxO1 and Adv-Empty vectors were subjected to H₂O₂ treatment, followed by real-time qRT-PCR analysis of prosurvival and proapoptotic gene expression. Data were from 3–5 independent experiments. *, P < .05 and **, P < .001 vs control.