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FoxO1 gain of function in the pancreas causes glucose intolerance, polycystic pancreas, and islet hypervascularization.

Kikuchi O, Kobayashi M, Amano K, Sasaki T, Kitazumi T, Kim HJ, Lee YS, Yokota-Hashimoto H, Kitamura YI, Kitamura T - PLoS ONE (2012)

Bottom Line: FoxO1 is a downstream transcription factor of insulin/IGF-1 signaling.We found FoxO1 binds to the VEGF-A promoter and regulates VEGF-A transcription in β cells.We propose that dysregulation of FoxO1 activity in the pancreas could account for the development of diabetes and pancreatic cysts.

View Article: PubMed Central - PubMed

Affiliation: Metabolic Signal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan.

ABSTRACT
Genetic studies revealed that the ablation of insulin/IGF-1 signaling in the pancreas causes diabetes. FoxO1 is a downstream transcription factor of insulin/IGF-1 signaling. We previously reported that FoxO1 haploinsufficiency restored β cell mass and rescued diabetes in IRS2 knockout mice. However, it is still unclear whether FoxO1 dysregulation in the pancreas could be the cause of diabetes. To test this hypothesis, we generated transgenic mice overexpressing constitutively active FoxO1 specifically in the pancreas (TG). TG mice had impaired glucose tolerance and some of them indeed developed diabetes due to the reduction of β cell mass, which is associated with decreased Pdx1 and MafA in β cells. We also observed increased proliferation of pancreatic duct epithelial cells in TG mice and some mice developed a polycystic pancreas as they aged. Furthermore, TG mice exhibited islet hypervascularities due to increased VEGF-A expression in β cells. We found FoxO1 binds to the VEGF-A promoter and regulates VEGF-A transcription in β cells. We propose that dysregulation of FoxO1 activity in the pancreas could account for the development of diabetes and pancreatic cysts.

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Related in: MedlinePlus

Reduced β cell mass and decreased expression of Pdx1 and MafA in TG mice.(A) Double immunohistochemistry with anti-insulin and anti-glucagon antibodies were conducted in pancreatic sections from TG and control mice. Representative images of low (upper panels) and high (lower panels) magnifications are shown. (B) The % area of β cells (left panel) or α cells (right panel) vs. whole pancreatic area was scored as described in Methods. (C and D) Double immunostaining of insulin with Pdx1 (C) or MafA (D) were conducted in pancreatic sections from TG and control mice. Arrows indicate individual cysts in TG pancreas. Representative images are shown. (E) Islets were isolated from TG and control mice and used for analyses by real time RT-PCR. In each experiment (A–E), at least six male and six female mice of each genotype and ten sections per mouse were analyzed. All the results were normalized using GAPDH. There was no significant difference between male and female mice. Data represent mean ± SEM. An asterisk indicates P<0.05 by ANOVA.
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pone-0032249-g002: Reduced β cell mass and decreased expression of Pdx1 and MafA in TG mice.(A) Double immunohistochemistry with anti-insulin and anti-glucagon antibodies were conducted in pancreatic sections from TG and control mice. Representative images of low (upper panels) and high (lower panels) magnifications are shown. (B) The % area of β cells (left panel) or α cells (right panel) vs. whole pancreatic area was scored as described in Methods. (C and D) Double immunostaining of insulin with Pdx1 (C) or MafA (D) were conducted in pancreatic sections from TG and control mice. Arrows indicate individual cysts in TG pancreas. Representative images are shown. (E) Islets were isolated from TG and control mice and used for analyses by real time RT-PCR. In each experiment (A–E), at least six male and six female mice of each genotype and ten sections per mouse were analyzed. All the results were normalized using GAPDH. There was no significant difference between male and female mice. Data represent mean ± SEM. An asterisk indicates P<0.05 by ANOVA.

Mentions: We generated transgenic mice overexpressing constitutively active FoxO1 specifically in the pancreas, using a transgene encoding a 4.5 kb Pdx1 promoter [17] followed by the cDNA of a constitutively nuclear mutant of FoxO1 (FoxO1-ADA) [19]. In this system, the Pdx1 promoter could be inhibited by its FoxO1 transgene product, however we confirmed that more than 90% of pancreatic cells expressed FoxO1-ADA in these mice [15]. One possibility is that because Pdx1 promoter is in fact inhibited by FoxO1-ADA, the level of transgene expression is only twofold higher than endogenous gene [15]. Another possibility is that the inhibition of endogenous Pdx1 expression is not necessarily a result of a direct effect of FoxO1, but it can be secondary to increased mRNA degradation or to promoter elements located outside the region we used to make transgenic mouse. Though Pdx1 is known to express in the brain as well as pancreas, FoxO1-ADA expression was observed neither in the brain nor in the other organs except pancreas of TG mice (data not shown). Although we already reported the phenotype of pancreatic morphology during pancreas development in TG mice [15], the other phenotypes in adult TG mice, such as metabolic parameters and pancreas architecture remained unsolved. Therefore, we first examined blood glucose levels at 8 weeks of age from both fasted and ad-lib fed mice. As seen in Fig. 1A, around 30% of male TG mice showed hyperglycemia in both fasted and fed conditions. Interestingly, none of the female TG mice developed hyperglycemia. Plasma insulin secretion in response to intraperitoneal glucose injection was blunted in male diabetic TG mice, indicating that hyperglycemia was caused by impaired insulin secretion in these mice (Fig. 1B). However, around 70% of male and all female TG mice showed almost normal blood glucose levels (Fig. 1A); therefore, we next performed intraperitoneal glucose tolerance tests (IPGTT) in the TG mice that had normal blood glucose levels in fasted and fed states at 12 weeks of age. IPGTT revealed that male, but not female, TG mice had impaired glucose tolerance; although, statistical significance was seen only at 30 and 60 min (Fig. 1C). There was no difference in the average body weight between TG and control mice (TG; 24.3±1.1 g, control; 24.5±1.4 g). To rule out the possibility of insulin resistance in TG mice, we performed insulin tolerance test (ITT). There was no difference in ITT results between TG and control mice in both male and female (Fig. 1D), indicating that impaired glucose tolerance was not caused by the peripheral insulin resistance in TG mice. We also measured plasma insulin and glucagon levels in male TG mice. As shown in Fig. 1E, plasma insulin levels were significantly lower in TG than control mice in both fasted and fed conditions, which is consistent with the impaired glucose responsive insulin secretion in male TG mice (Fig. 1B). Interestingly, plasma glucagon levels were higher in TG than control mice in the fed conditions (Fig. 1E). Although the mechanism is unclear, enhanced glucagon secretion may deteriorate the glucose intolerance in TG mice. To further evaluate the impaired insulin secretion in TG mice, we isolated islets from TG and control mice and performed glucose stimulated insulin secretion (GSIS) assays. Because small islets in TG mice have lower β cell/α cell ratio than in the control mice (as shown in Fig. 2A), we used the size-matched large islets between TG and control mice, in which β cell mass should be comparable. Consistent with the data of plasma insulin levels, TG islets have decreased glucose-responsive insulin secretion (Fig. 1F). We next started feeding a high fat high sucrose diet (HFHSD) to the TG mice whose blood glucose levels were normal, at least until 4 weeks of age. After HFHSD feeding for 8 weeks, we measured blood glucose levels and found the average blood glucose level in male TG mice was significantly higher than in control mice, and these mice developed overt diabetes (Fig. 1G). IPGTT revealed that not only male, but also female TG mice exhibited significantly impaired glucose tolerance under a HFHSD (Fig. 1H).


FoxO1 gain of function in the pancreas causes glucose intolerance, polycystic pancreas, and islet hypervascularization.

Kikuchi O, Kobayashi M, Amano K, Sasaki T, Kitazumi T, Kim HJ, Lee YS, Yokota-Hashimoto H, Kitamura YI, Kitamura T - PLoS ONE (2012)

Reduced β cell mass and decreased expression of Pdx1 and MafA in TG mice.(A) Double immunohistochemistry with anti-insulin and anti-glucagon antibodies were conducted in pancreatic sections from TG and control mice. Representative images of low (upper panels) and high (lower panels) magnifications are shown. (B) The % area of β cells (left panel) or α cells (right panel) vs. whole pancreatic area was scored as described in Methods. (C and D) Double immunostaining of insulin with Pdx1 (C) or MafA (D) were conducted in pancreatic sections from TG and control mice. Arrows indicate individual cysts in TG pancreas. Representative images are shown. (E) Islets were isolated from TG and control mice and used for analyses by real time RT-PCR. In each experiment (A–E), at least six male and six female mice of each genotype and ten sections per mouse were analyzed. All the results were normalized using GAPDH. There was no significant difference between male and female mice. Data represent mean ± SEM. An asterisk indicates P<0.05 by ANOVA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285669&req=5

pone-0032249-g002: Reduced β cell mass and decreased expression of Pdx1 and MafA in TG mice.(A) Double immunohistochemistry with anti-insulin and anti-glucagon antibodies were conducted in pancreatic sections from TG and control mice. Representative images of low (upper panels) and high (lower panels) magnifications are shown. (B) The % area of β cells (left panel) or α cells (right panel) vs. whole pancreatic area was scored as described in Methods. (C and D) Double immunostaining of insulin with Pdx1 (C) or MafA (D) were conducted in pancreatic sections from TG and control mice. Arrows indicate individual cysts in TG pancreas. Representative images are shown. (E) Islets were isolated from TG and control mice and used for analyses by real time RT-PCR. In each experiment (A–E), at least six male and six female mice of each genotype and ten sections per mouse were analyzed. All the results were normalized using GAPDH. There was no significant difference between male and female mice. Data represent mean ± SEM. An asterisk indicates P<0.05 by ANOVA.
Mentions: We generated transgenic mice overexpressing constitutively active FoxO1 specifically in the pancreas, using a transgene encoding a 4.5 kb Pdx1 promoter [17] followed by the cDNA of a constitutively nuclear mutant of FoxO1 (FoxO1-ADA) [19]. In this system, the Pdx1 promoter could be inhibited by its FoxO1 transgene product, however we confirmed that more than 90% of pancreatic cells expressed FoxO1-ADA in these mice [15]. One possibility is that because Pdx1 promoter is in fact inhibited by FoxO1-ADA, the level of transgene expression is only twofold higher than endogenous gene [15]. Another possibility is that the inhibition of endogenous Pdx1 expression is not necessarily a result of a direct effect of FoxO1, but it can be secondary to increased mRNA degradation or to promoter elements located outside the region we used to make transgenic mouse. Though Pdx1 is known to express in the brain as well as pancreas, FoxO1-ADA expression was observed neither in the brain nor in the other organs except pancreas of TG mice (data not shown). Although we already reported the phenotype of pancreatic morphology during pancreas development in TG mice [15], the other phenotypes in adult TG mice, such as metabolic parameters and pancreas architecture remained unsolved. Therefore, we first examined blood glucose levels at 8 weeks of age from both fasted and ad-lib fed mice. As seen in Fig. 1A, around 30% of male TG mice showed hyperglycemia in both fasted and fed conditions. Interestingly, none of the female TG mice developed hyperglycemia. Plasma insulin secretion in response to intraperitoneal glucose injection was blunted in male diabetic TG mice, indicating that hyperglycemia was caused by impaired insulin secretion in these mice (Fig. 1B). However, around 70% of male and all female TG mice showed almost normal blood glucose levels (Fig. 1A); therefore, we next performed intraperitoneal glucose tolerance tests (IPGTT) in the TG mice that had normal blood glucose levels in fasted and fed states at 12 weeks of age. IPGTT revealed that male, but not female, TG mice had impaired glucose tolerance; although, statistical significance was seen only at 30 and 60 min (Fig. 1C). There was no difference in the average body weight between TG and control mice (TG; 24.3±1.1 g, control; 24.5±1.4 g). To rule out the possibility of insulin resistance in TG mice, we performed insulin tolerance test (ITT). There was no difference in ITT results between TG and control mice in both male and female (Fig. 1D), indicating that impaired glucose tolerance was not caused by the peripheral insulin resistance in TG mice. We also measured plasma insulin and glucagon levels in male TG mice. As shown in Fig. 1E, plasma insulin levels were significantly lower in TG than control mice in both fasted and fed conditions, which is consistent with the impaired glucose responsive insulin secretion in male TG mice (Fig. 1B). Interestingly, plasma glucagon levels were higher in TG than control mice in the fed conditions (Fig. 1E). Although the mechanism is unclear, enhanced glucagon secretion may deteriorate the glucose intolerance in TG mice. To further evaluate the impaired insulin secretion in TG mice, we isolated islets from TG and control mice and performed glucose stimulated insulin secretion (GSIS) assays. Because small islets in TG mice have lower β cell/α cell ratio than in the control mice (as shown in Fig. 2A), we used the size-matched large islets between TG and control mice, in which β cell mass should be comparable. Consistent with the data of plasma insulin levels, TG islets have decreased glucose-responsive insulin secretion (Fig. 1F). We next started feeding a high fat high sucrose diet (HFHSD) to the TG mice whose blood glucose levels were normal, at least until 4 weeks of age. After HFHSD feeding for 8 weeks, we measured blood glucose levels and found the average blood glucose level in male TG mice was significantly higher than in control mice, and these mice developed overt diabetes (Fig. 1G). IPGTT revealed that not only male, but also female TG mice exhibited significantly impaired glucose tolerance under a HFHSD (Fig. 1H).

Bottom Line: FoxO1 is a downstream transcription factor of insulin/IGF-1 signaling.We found FoxO1 binds to the VEGF-A promoter and regulates VEGF-A transcription in β cells.We propose that dysregulation of FoxO1 activity in the pancreas could account for the development of diabetes and pancreatic cysts.

View Article: PubMed Central - PubMed

Affiliation: Metabolic Signal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan.

ABSTRACT
Genetic studies revealed that the ablation of insulin/IGF-1 signaling in the pancreas causes diabetes. FoxO1 is a downstream transcription factor of insulin/IGF-1 signaling. We previously reported that FoxO1 haploinsufficiency restored β cell mass and rescued diabetes in IRS2 knockout mice. However, it is still unclear whether FoxO1 dysregulation in the pancreas could be the cause of diabetes. To test this hypothesis, we generated transgenic mice overexpressing constitutively active FoxO1 specifically in the pancreas (TG). TG mice had impaired glucose tolerance and some of them indeed developed diabetes due to the reduction of β cell mass, which is associated with decreased Pdx1 and MafA in β cells. We also observed increased proliferation of pancreatic duct epithelial cells in TG mice and some mice developed a polycystic pancreas as they aged. Furthermore, TG mice exhibited islet hypervascularities due to increased VEGF-A expression in β cells. We found FoxO1 binds to the VEGF-A promoter and regulates VEGF-A transcription in β cells. We propose that dysregulation of FoxO1 activity in the pancreas could account for the development of diabetes and pancreatic cysts.

Show MeSH
Related in: MedlinePlus