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CREB mediates the insulinotropic and anti-apoptotic effects of GLP-1 signaling in adult mouse β-cells.

Shin S, Le Lay J, Everett LJ, Gupta R, Rafiq K, Kaestner KH - Mol Metab (2014)

Bottom Line: Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis.Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in β-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling.In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT

Objective: Glucagon-like peptide-1 (GLP-1) plays a major role in pancreatic β-cell function and survival by increasing cytoplasmic cAMP levels, which are thought to affect transcription through activation of the basic leucine zipper (bZIP) transcription factor CREB. Here, we test CREB function in the adult β-cell through inducible gene deletion.

Methods: We employed cell type-specific and inducible gene ablation to determine CREB function in pancreatic β-cells in mice.

Results: By ablating CREB acutely in mature β-cells in tamoxifen-treated Creb (loxP/loxP);Pdx1-CreERT2 mice, we show that CREB has little impact on β-cell turnover, in contrast to what had been postulated previously. Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis. Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in β-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling.

Conclusions: In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass. However, we reveal an important role for CREB in the β-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

No MeSH data available.


Related in: MedlinePlus

β-cell Mass is not affected in high-fat diet-fed CrebloxP/loxP;Pdx1CreERT2 mice. (A) Quantification of β-cell mass in control (L/L) and mutant (L/L, CreERT2) mice (n = 4–5). Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. (B) Quantitative RT-PCR to measure relative mRNA levels of insulin (Ins1 and Ins2) and glucagon (Gcg) in islets isolated from control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet (n = 10–14). (C) Quantitative RT-PCR to measure relative mRNA levels of presumed Creb target genes that may be associated with β-cell turnover in islets isolated from tamoxifen-treated control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet. Transcript levels in control mice were assigned an arbitrary value of 1.0 for comparison (n = 6). All values are expressed as mean + SEM. (D) Immunostaining of pancreas sections demonstrating that replication of islet β-cells is unaffected by Creb deficiency. Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. Insulin-positive β-cells (green) and 5-bromo-2′-deoxyuridine (BrdU)-positive cells (red) are shown. Arrowheads indicate β-cells labeled with BrdU. BrdU were administered in drinking water (1 g/L) for one week.
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fig2: β-cell Mass is not affected in high-fat diet-fed CrebloxP/loxP;Pdx1CreERT2 mice. (A) Quantification of β-cell mass in control (L/L) and mutant (L/L, CreERT2) mice (n = 4–5). Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. (B) Quantitative RT-PCR to measure relative mRNA levels of insulin (Ins1 and Ins2) and glucagon (Gcg) in islets isolated from control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet (n = 10–14). (C) Quantitative RT-PCR to measure relative mRNA levels of presumed Creb target genes that may be associated with β-cell turnover in islets isolated from tamoxifen-treated control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet. Transcript levels in control mice were assigned an arbitrary value of 1.0 for comparison (n = 6). All values are expressed as mean + SEM. (D) Immunostaining of pancreas sections demonstrating that replication of islet β-cells is unaffected by Creb deficiency. Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. Insulin-positive β-cells (green) and 5-bromo-2′-deoxyuridine (BrdU)-positive cells (red) are shown. Arrowheads indicate β-cells labeled with BrdU. BrdU were administered in drinking water (1 g/L) for one week.

Mentions: Prior studies using dominant-negative approaches have suggested that CREB plays an important role in maintaining β-cell mass [18,19]. Jhala and colleagues observed massive β-cell apoptosis in their transgenic mouse model and attributed that to downregulation of insulin receptor substrate-2 (IRS-2) [18]. In contrast, Inada and colleagues found no evidence for increased apoptosis in their transgenic model of ICER overexpression, but rather observed reduced β-cell replication, which they ascribed to attenuated cyclin A2 expression [19]. Therefore, we assessed β-cell mass, as well as expression of these two proposed targets of CREB activity in β-cells, in our genetic model of CREB ablation. Surprisingly, β-cell mass was not altered in high-fat diet-fed female mutant mice compared to controls (Figure 2A). Likewise, mRNA levels of insulin and glucagon did not change following deletion of CREB (Figure 2B), indicating that CREB deficiency in mature β-cell does not affect β-cell mass or hormone gene expression. Furthermore, mRNA levels of both the insulin receptor substrate 2 (Irs2) and cyclin A2 (Ccna2) were unaffected in CREB-deficient islets (Figure 2C). In addition, gene expression of Irs1, cyclin D1 (Ccnd1), and B-cell lymphoma 2 (Bcl2), three additional proposed CREB targets that could have an impact on β-cell turnover, were similarly unchanged in the islets of CREB mutants (Figure 2C). Finally, proliferation rates of β-cells detected by continuous 5-bromo-2′-deoxyuridine (BrdU) labeling remained unaltered by CREB deficiency (Figure 2D). These data strongly suggest that the previously reported phenotypes in the dominant-negative transgenic mouse models were the result of off-target effects (see Section 4).


CREB mediates the insulinotropic and anti-apoptotic effects of GLP-1 signaling in adult mouse β-cells.

Shin S, Le Lay J, Everett LJ, Gupta R, Rafiq K, Kaestner KH - Mol Metab (2014)

β-cell Mass is not affected in high-fat diet-fed CrebloxP/loxP;Pdx1CreERT2 mice. (A) Quantification of β-cell mass in control (L/L) and mutant (L/L, CreERT2) mice (n = 4–5). Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. (B) Quantitative RT-PCR to measure relative mRNA levels of insulin (Ins1 and Ins2) and glucagon (Gcg) in islets isolated from control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet (n = 10–14). (C) Quantitative RT-PCR to measure relative mRNA levels of presumed Creb target genes that may be associated with β-cell turnover in islets isolated from tamoxifen-treated control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet. Transcript levels in control mice were assigned an arbitrary value of 1.0 for comparison (n = 6). All values are expressed as mean + SEM. (D) Immunostaining of pancreas sections demonstrating that replication of islet β-cells is unaffected by Creb deficiency. Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. Insulin-positive β-cells (green) and 5-bromo-2′-deoxyuridine (BrdU)-positive cells (red) are shown. Arrowheads indicate β-cells labeled with BrdU. BrdU were administered in drinking water (1 g/L) for one week.
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Related In: Results  -  Collection

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fig2: β-cell Mass is not affected in high-fat diet-fed CrebloxP/loxP;Pdx1CreERT2 mice. (A) Quantification of β-cell mass in control (L/L) and mutant (L/L, CreERT2) mice (n = 4–5). Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. (B) Quantitative RT-PCR to measure relative mRNA levels of insulin (Ins1 and Ins2) and glucagon (Gcg) in islets isolated from control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet (n = 10–14). (C) Quantitative RT-PCR to measure relative mRNA levels of presumed Creb target genes that may be associated with β-cell turnover in islets isolated from tamoxifen-treated control (L/L) and mutant (L/L, CreERT2) female mice fed a high-fat diet. Transcript levels in control mice were assigned an arbitrary value of 1.0 for comparison (n = 6). All values are expressed as mean + SEM. (D) Immunostaining of pancreas sections demonstrating that replication of islet β-cells is unaffected by Creb deficiency. Female mice were fed a high-fat diet for 10–11 weeks starting when the mice were 4–5 weeks old. Insulin-positive β-cells (green) and 5-bromo-2′-deoxyuridine (BrdU)-positive cells (red) are shown. Arrowheads indicate β-cells labeled with BrdU. BrdU were administered in drinking water (1 g/L) for one week.
Mentions: Prior studies using dominant-negative approaches have suggested that CREB plays an important role in maintaining β-cell mass [18,19]. Jhala and colleagues observed massive β-cell apoptosis in their transgenic mouse model and attributed that to downregulation of insulin receptor substrate-2 (IRS-2) [18]. In contrast, Inada and colleagues found no evidence for increased apoptosis in their transgenic model of ICER overexpression, but rather observed reduced β-cell replication, which they ascribed to attenuated cyclin A2 expression [19]. Therefore, we assessed β-cell mass, as well as expression of these two proposed targets of CREB activity in β-cells, in our genetic model of CREB ablation. Surprisingly, β-cell mass was not altered in high-fat diet-fed female mutant mice compared to controls (Figure 2A). Likewise, mRNA levels of insulin and glucagon did not change following deletion of CREB (Figure 2B), indicating that CREB deficiency in mature β-cell does not affect β-cell mass or hormone gene expression. Furthermore, mRNA levels of both the insulin receptor substrate 2 (Irs2) and cyclin A2 (Ccna2) were unaffected in CREB-deficient islets (Figure 2C). In addition, gene expression of Irs1, cyclin D1 (Ccnd1), and B-cell lymphoma 2 (Bcl2), three additional proposed CREB targets that could have an impact on β-cell turnover, were similarly unchanged in the islets of CREB mutants (Figure 2C). Finally, proliferation rates of β-cells detected by continuous 5-bromo-2′-deoxyuridine (BrdU) labeling remained unaltered by CREB deficiency (Figure 2D). These data strongly suggest that the previously reported phenotypes in the dominant-negative transgenic mouse models were the result of off-target effects (see Section 4).

Bottom Line: Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis.Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in β-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling.In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT

Objective: Glucagon-like peptide-1 (GLP-1) plays a major role in pancreatic β-cell function and survival by increasing cytoplasmic cAMP levels, which are thought to affect transcription through activation of the basic leucine zipper (bZIP) transcription factor CREB. Here, we test CREB function in the adult β-cell through inducible gene deletion.

Methods: We employed cell type-specific and inducible gene ablation to determine CREB function in pancreatic β-cells in mice.

Results: By ablating CREB acutely in mature β-cells in tamoxifen-treated Creb (loxP/loxP);Pdx1-CreERT2 mice, we show that CREB has little impact on β-cell turnover, in contrast to what had been postulated previously. Rather, CREB is required for GLP-1 to elicit its full effects on stimulating glucose-induced insulin secretion and protection from cytokine-induced apoptosis. Mechanistically, we find that CREB regulates expression of the pro-apoptotic gene p21 (Cdkn1a) in β-cells, thus demonstrating that CREB is essential to mediating this critical aspect of GLP-1 receptor signaling.

Conclusions: In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass. However, we reveal an important role for CREB in the β-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

No MeSH data available.


Related in: MedlinePlus