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Physiologic and pharmacologic modulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in beta-cells by peroxisome proliferator-activated receptor (PPAR)-gamma signaling: possible mechanism for the GIP resistance in type 2 diabetes.

Gupta D, Peshavaria M, Monga N, Jetton TL, Leahy JL - Diabetes (2010)

Bottom Line: In vitro studies of INS-1 cells confirmed that PPAR-gamma binds to the putative PPRE sequence and regulates GIP-R transcription.In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARgamma(-/-) mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress beta-cell PPARgamma.Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in beta-cells by PPARgamma signaling.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes, and Metabolism and the Department of Medicine, University of Vermont, Burlington, Vermont, USA.

ABSTRACT

Objective: We previously showed that peroxisome proliferator-activated receptor (PPAR)-gamma in beta-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARgamma in GIP-R transcription.

Research design and methods: Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARgamma knockout mice (PANC PPARgamma(-/-)), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone.

Results: In vitro studies of INS-1 cells confirmed that PPAR-gamma binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARgamma(-/-) mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress beta-cell PPARgamma. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARgamma and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px.

Conclusions: Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in beta-cells by PPARgamma signaling. Also disruption of this signaling pathway may account for the lowered beta-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

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Related in: MedlinePlus

Experiments in INS-1 cells. A: Chromatin immunoprecipitation assay to assess PPARγ binding to the putative PPRE on the GIP-R gene. Chromatin fragments (300–500 bp length) were generated. Representative gel showing chromatin preparations from two separate experiments immunoprecipitated with rabbit polyclonal anti-PPARγ (lanes 2 and 3) or the negative control nonimmune serum (lanes 4 and 5). Lane 1 is nonimmunoprecipitated DNA. Results show the expected 213-bp PCR product with the anti-PPARγ and input DNA, but not the control serum. B: PPARγ siRNA. INS-1 cells underwent transfections with siRNA duplexes against the rat PPARγ gene or scrambled siRNA duplexes. Cells were cultured with media that contained 10 μmol/l troglitazone or DMSO for 72 h, and the isolated RNA was assessed for GIP-R mRNA levels by RT-PCR. Representative gels show scrambled siRNA cells cultured with DMSO (lane 1), scrambled siRNA cells cultured with troglitazone (lane 2), siRNA cells cultured with troglitazone (lane 3), and siRNA cells cultured with DMSO (lane 4). Cyclophilin B mRNA was used as an internal control. C and D: Luciferase reporter transcription assay. INS-1 cells were transfected with wild-type or mutated pTAL-PPRE-rat GIP-R vectors, and 24 h post-transfection, the cells were treated with 10 μmol/l troglitazone or DMSO for 24 h. WT = wild-type rat GIP-R PPRE (CCCATG-G-AGGTCA). Mut-1 = mutation of the 5′ DR1 half-site of the rat GIP-R PPRE (AAAATA-G-AGGTCA). Mut-2 = mutation of the 3′ DR2 half-site of the rat GIP-R PPRE (CCCATG-G-ATTTTA). C shows the relative basal luciferase activity (DMSO-treated cells) compared with the wild-type rat GIP-R PPRE construct as means ± SEM of three separate experiments. D shows the troglitazone treatment effect on luciferase reporter activity of the wild-type and mutated GIP-R PPREs. *P < 0.015 vs. DMSO-cultured wild-type GIP-R PPRE cells. #P < 0.001 vs. troglitazone cultured wild-type GIP-R PPRE cells.
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Figure 1: Experiments in INS-1 cells. A: Chromatin immunoprecipitation assay to assess PPARγ binding to the putative PPRE on the GIP-R gene. Chromatin fragments (300–500 bp length) were generated. Representative gel showing chromatin preparations from two separate experiments immunoprecipitated with rabbit polyclonal anti-PPARγ (lanes 2 and 3) or the negative control nonimmune serum (lanes 4 and 5). Lane 1 is nonimmunoprecipitated DNA. Results show the expected 213-bp PCR product with the anti-PPARγ and input DNA, but not the control serum. B: PPARγ siRNA. INS-1 cells underwent transfections with siRNA duplexes against the rat PPARγ gene or scrambled siRNA duplexes. Cells were cultured with media that contained 10 μmol/l troglitazone or DMSO for 72 h, and the isolated RNA was assessed for GIP-R mRNA levels by RT-PCR. Representative gels show scrambled siRNA cells cultured with DMSO (lane 1), scrambled siRNA cells cultured with troglitazone (lane 2), siRNA cells cultured with troglitazone (lane 3), and siRNA cells cultured with DMSO (lane 4). Cyclophilin B mRNA was used as an internal control. C and D: Luciferase reporter transcription assay. INS-1 cells were transfected with wild-type or mutated pTAL-PPRE-rat GIP-R vectors, and 24 h post-transfection, the cells were treated with 10 μmol/l troglitazone or DMSO for 24 h. WT = wild-type rat GIP-R PPRE (CCCATG-G-AGGTCA). Mut-1 = mutation of the 5′ DR1 half-site of the rat GIP-R PPRE (AAAATA-G-AGGTCA). Mut-2 = mutation of the 3′ DR2 half-site of the rat GIP-R PPRE (CCCATG-G-ATTTTA). C shows the relative basal luciferase activity (DMSO-treated cells) compared with the wild-type rat GIP-R PPRE construct as means ± SEM of three separate experiments. D shows the troglitazone treatment effect on luciferase reporter activity of the wild-type and mutated GIP-R PPREs. *P < 0.015 vs. DMSO-cultured wild-type GIP-R PPRE cells. #P < 0.001 vs. troglitazone cultured wild-type GIP-R PPRE cells.

Mentions: Studies were performed in rat-derived INS-1 cells to confirm functionality of this putative PPRE. PPARγ binding was determined with the chromatin immunoprecipitation assay. Flanking primer pairs for a 213-bp PCR product that included the GIP-R PPRE (schema in supplemental Fig. 2A) generated the correct-sized PCR band with input DNA and PPARγ antibody–precipitated DNA, whereas only faint bands were observed with nonimmune serum (Fig. 1A). Representative negative and positive controls are shown in supplemental Fig. 2.


Physiologic and pharmacologic modulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in beta-cells by peroxisome proliferator-activated receptor (PPAR)-gamma signaling: possible mechanism for the GIP resistance in type 2 diabetes.

Gupta D, Peshavaria M, Monga N, Jetton TL, Leahy JL - Diabetes (2010)

Experiments in INS-1 cells. A: Chromatin immunoprecipitation assay to assess PPARγ binding to the putative PPRE on the GIP-R gene. Chromatin fragments (300–500 bp length) were generated. Representative gel showing chromatin preparations from two separate experiments immunoprecipitated with rabbit polyclonal anti-PPARγ (lanes 2 and 3) or the negative control nonimmune serum (lanes 4 and 5). Lane 1 is nonimmunoprecipitated DNA. Results show the expected 213-bp PCR product with the anti-PPARγ and input DNA, but not the control serum. B: PPARγ siRNA. INS-1 cells underwent transfections with siRNA duplexes against the rat PPARγ gene or scrambled siRNA duplexes. Cells were cultured with media that contained 10 μmol/l troglitazone or DMSO for 72 h, and the isolated RNA was assessed for GIP-R mRNA levels by RT-PCR. Representative gels show scrambled siRNA cells cultured with DMSO (lane 1), scrambled siRNA cells cultured with troglitazone (lane 2), siRNA cells cultured with troglitazone (lane 3), and siRNA cells cultured with DMSO (lane 4). Cyclophilin B mRNA was used as an internal control. C and D: Luciferase reporter transcription assay. INS-1 cells were transfected with wild-type or mutated pTAL-PPRE-rat GIP-R vectors, and 24 h post-transfection, the cells were treated with 10 μmol/l troglitazone or DMSO for 24 h. WT = wild-type rat GIP-R PPRE (CCCATG-G-AGGTCA). Mut-1 = mutation of the 5′ DR1 half-site of the rat GIP-R PPRE (AAAATA-G-AGGTCA). Mut-2 = mutation of the 3′ DR2 half-site of the rat GIP-R PPRE (CCCATG-G-ATTTTA). C shows the relative basal luciferase activity (DMSO-treated cells) compared with the wild-type rat GIP-R PPRE construct as means ± SEM of three separate experiments. D shows the troglitazone treatment effect on luciferase reporter activity of the wild-type and mutated GIP-R PPREs. *P < 0.015 vs. DMSO-cultured wild-type GIP-R PPRE cells. #P < 0.001 vs. troglitazone cultured wild-type GIP-R PPRE cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2874705&req=5

Figure 1: Experiments in INS-1 cells. A: Chromatin immunoprecipitation assay to assess PPARγ binding to the putative PPRE on the GIP-R gene. Chromatin fragments (300–500 bp length) were generated. Representative gel showing chromatin preparations from two separate experiments immunoprecipitated with rabbit polyclonal anti-PPARγ (lanes 2 and 3) or the negative control nonimmune serum (lanes 4 and 5). Lane 1 is nonimmunoprecipitated DNA. Results show the expected 213-bp PCR product with the anti-PPARγ and input DNA, but not the control serum. B: PPARγ siRNA. INS-1 cells underwent transfections with siRNA duplexes against the rat PPARγ gene or scrambled siRNA duplexes. Cells were cultured with media that contained 10 μmol/l troglitazone or DMSO for 72 h, and the isolated RNA was assessed for GIP-R mRNA levels by RT-PCR. Representative gels show scrambled siRNA cells cultured with DMSO (lane 1), scrambled siRNA cells cultured with troglitazone (lane 2), siRNA cells cultured with troglitazone (lane 3), and siRNA cells cultured with DMSO (lane 4). Cyclophilin B mRNA was used as an internal control. C and D: Luciferase reporter transcription assay. INS-1 cells were transfected with wild-type or mutated pTAL-PPRE-rat GIP-R vectors, and 24 h post-transfection, the cells were treated with 10 μmol/l troglitazone or DMSO for 24 h. WT = wild-type rat GIP-R PPRE (CCCATG-G-AGGTCA). Mut-1 = mutation of the 5′ DR1 half-site of the rat GIP-R PPRE (AAAATA-G-AGGTCA). Mut-2 = mutation of the 3′ DR2 half-site of the rat GIP-R PPRE (CCCATG-G-ATTTTA). C shows the relative basal luciferase activity (DMSO-treated cells) compared with the wild-type rat GIP-R PPRE construct as means ± SEM of three separate experiments. D shows the troglitazone treatment effect on luciferase reporter activity of the wild-type and mutated GIP-R PPREs. *P < 0.015 vs. DMSO-cultured wild-type GIP-R PPRE cells. #P < 0.001 vs. troglitazone cultured wild-type GIP-R PPRE cells.
Mentions: Studies were performed in rat-derived INS-1 cells to confirm functionality of this putative PPRE. PPARγ binding was determined with the chromatin immunoprecipitation assay. Flanking primer pairs for a 213-bp PCR product that included the GIP-R PPRE (schema in supplemental Fig. 2A) generated the correct-sized PCR band with input DNA and PPARγ antibody–precipitated DNA, whereas only faint bands were observed with nonimmune serum (Fig. 1A). Representative negative and positive controls are shown in supplemental Fig. 2.

Bottom Line: In vitro studies of INS-1 cells confirmed that PPAR-gamma binds to the putative PPRE sequence and regulates GIP-R transcription.In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARgamma(-/-) mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress beta-cell PPARgamma.Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in beta-cells by PPARgamma signaling.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes, and Metabolism and the Department of Medicine, University of Vermont, Burlington, Vermont, USA.

ABSTRACT

Objective: We previously showed that peroxisome proliferator-activated receptor (PPAR)-gamma in beta-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARgamma in GIP-R transcription.

Research design and methods: Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARgamma knockout mice (PANC PPARgamma(-/-)), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone.

Results: In vitro studies of INS-1 cells confirmed that PPAR-gamma binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARgamma(-/-) mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress beta-cell PPARgamma. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARgamma and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px.

Conclusions: Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in beta-cells by PPARgamma signaling. Also disruption of this signaling pathway may account for the lowered beta-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Show MeSH
Related in: MedlinePlus