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Combination treatment of db/db mice with exendin-4 and gastrin preserves β-cell mass by stimulating β-cell growth and differentiation.

Tamaki M, Fujitani Y, Uchida T, Hirose T, Kawamori R, Watada H - J Diabetes Investig (2010)

Bottom Line: Immunohistochemistry, western blotting and RT-PCR assays were used to assess the biological effects of the two agents.   Two weeks of combination administration of exendin-4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β-cell mass in db/db mice.While gastrin had little biological effect on INS-1 β-cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin-producing cells.Co-stimulation with exendin-4 significantly enhanced gastrin-induced endocrine differentiation of AR42J precursor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Metabolism and Endocrinology.

ABSTRACT

Unlabelled: Aim/Introduction:  Preservation of β-cell mass is crucial for maintaining long-term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of β-cell mass. Here we tested the effects of gastrin and exendin-4 in type 2 diabetic animals.

Materials and methods:   The effects of exendin-4 and gastrin on β-cell function and mass were examined in 8-week-old db/db mice. INS-1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT-PCR assays were used to assess the biological effects of the two agents.

Results:   Two weeks of combination administration of exendin-4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β-cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly-shaped small islets and single insulin-positive cells. While gastrin had little biological effect on INS-1 β-cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin-producing cells. Co-stimulation with exendin-4 significantly enhanced gastrin-induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in β-cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA.

Conclusions:   These results suggest that combination treatment of db/db mice with exendin-4 and gastrin preserves β-cell mass by stimulating β-cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.00044.x, 2010).

No MeSH data available.


Related in: MedlinePlus

 (a–d,f) Insulin expression in pancreatic precursor cell line AR42J treated with exendin‐4 and/or gastrin. AR42J cells treated for 72 h with exendin‐4 (1 nmol/L) and/or gastrin (10 nmol/L) were stained with anti‐insulin antibody (green) and DAPI (blue). (a) Untreated AR42J cells (C). (b) Gastrin‐treated AR42J cells (G). (c) Exendin‐4‐treated AR42J cells (E). (d,f) AR42J cells treated with exendin‐4 plus gastrin (E&G). (e) INS‐1 insulinoma cells as a positive control. (g) Percentage of insulin‐positive cells in each group. Data are mean ± SEM (n = 3). *P < 0.05 vs C group, §P < 0.05 vs E&G group. (h) Western blot analysis was carried out to assess insulin content of AR42J cells treated with exendin‐4 and/or gastrin for 3 days. INS‐1 insulinoma cells were used as a positive control. Representative data of three experiments with similar results.
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f4:  (a–d,f) Insulin expression in pancreatic precursor cell line AR42J treated with exendin‐4 and/or gastrin. AR42J cells treated for 72 h with exendin‐4 (1 nmol/L) and/or gastrin (10 nmol/L) were stained with anti‐insulin antibody (green) and DAPI (blue). (a) Untreated AR42J cells (C). (b) Gastrin‐treated AR42J cells (G). (c) Exendin‐4‐treated AR42J cells (E). (d,f) AR42J cells treated with exendin‐4 plus gastrin (E&G). (e) INS‐1 insulinoma cells as a positive control. (g) Percentage of insulin‐positive cells in each group. Data are mean ± SEM (n = 3). *P < 0.05 vs C group, §P < 0.05 vs E&G group. (h) Western blot analysis was carried out to assess insulin content of AR42J cells treated with exendin‐4 and/or gastrin for 3 days. INS‐1 insulinoma cells were used as a positive control. Representative data of three experiments with similar results.

Mentions: Previous studies reported that treatment of AR42J cells, a tumor cell line showing pancreatic progenitor‐like properties, with GLP‐1/exendin‐4 induced insulin‐producing cells16,17. In preliminary experiments, the optimal concentrations of exendin‐4 and gastrin for induction of efficient phosphorylation of p42/44 MAPK in AR42J were found to be 1 and 10 nmol/L, respectively (data not shown). To assess whether or not exendin‐4 and/or gastrin can generate insulin‐producing cells from AR42J progenitors, cells were treated with 1 nmol/L exendin‐4 and/or 10 nmol/L gastrin for various intervals followed by immunostaining with anti‐insulin antibody. Optimal induction of insulin‐positive cells was observed when AR42J cells were incubated for 3 days with E&G (Figure 4a–d). The proportion of insulin‐positive cells in the exendin‐4 or gastrin‐treated group was significantly higher than untreated control cells. Importantly, E&G co‐stimulation resulted in a significant increase in the proportion of insulin‐positive cells than monotherapy with exendin‐4 or gastrin. Western blot analysis further confirmed that insulin protein was indeed synthesized in AR42J cells cultured with exendin‐4 or gastrin and that insulin protein levels were synergistically increased by co‐stimulation with the two agents (Figure 4h).


Combination treatment of db/db mice with exendin-4 and gastrin preserves β-cell mass by stimulating β-cell growth and differentiation.

Tamaki M, Fujitani Y, Uchida T, Hirose T, Kawamori R, Watada H - J Diabetes Investig (2010)

 (a–d,f) Insulin expression in pancreatic precursor cell line AR42J treated with exendin‐4 and/or gastrin. AR42J cells treated for 72 h with exendin‐4 (1 nmol/L) and/or gastrin (10 nmol/L) were stained with anti‐insulin antibody (green) and DAPI (blue). (a) Untreated AR42J cells (C). (b) Gastrin‐treated AR42J cells (G). (c) Exendin‐4‐treated AR42J cells (E). (d,f) AR42J cells treated with exendin‐4 plus gastrin (E&G). (e) INS‐1 insulinoma cells as a positive control. (g) Percentage of insulin‐positive cells in each group. Data are mean ± SEM (n = 3). *P < 0.05 vs C group, §P < 0.05 vs E&G group. (h) Western blot analysis was carried out to assess insulin content of AR42J cells treated with exendin‐4 and/or gastrin for 3 days. INS‐1 insulinoma cells were used as a positive control. Representative data of three experiments with similar results.
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Related In: Results  -  Collection

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f4:  (a–d,f) Insulin expression in pancreatic precursor cell line AR42J treated with exendin‐4 and/or gastrin. AR42J cells treated for 72 h with exendin‐4 (1 nmol/L) and/or gastrin (10 nmol/L) were stained with anti‐insulin antibody (green) and DAPI (blue). (a) Untreated AR42J cells (C). (b) Gastrin‐treated AR42J cells (G). (c) Exendin‐4‐treated AR42J cells (E). (d,f) AR42J cells treated with exendin‐4 plus gastrin (E&G). (e) INS‐1 insulinoma cells as a positive control. (g) Percentage of insulin‐positive cells in each group. Data are mean ± SEM (n = 3). *P < 0.05 vs C group, §P < 0.05 vs E&G group. (h) Western blot analysis was carried out to assess insulin content of AR42J cells treated with exendin‐4 and/or gastrin for 3 days. INS‐1 insulinoma cells were used as a positive control. Representative data of three experiments with similar results.
Mentions: Previous studies reported that treatment of AR42J cells, a tumor cell line showing pancreatic progenitor‐like properties, with GLP‐1/exendin‐4 induced insulin‐producing cells16,17. In preliminary experiments, the optimal concentrations of exendin‐4 and gastrin for induction of efficient phosphorylation of p42/44 MAPK in AR42J were found to be 1 and 10 nmol/L, respectively (data not shown). To assess whether or not exendin‐4 and/or gastrin can generate insulin‐producing cells from AR42J progenitors, cells were treated with 1 nmol/L exendin‐4 and/or 10 nmol/L gastrin for various intervals followed by immunostaining with anti‐insulin antibody. Optimal induction of insulin‐positive cells was observed when AR42J cells were incubated for 3 days with E&G (Figure 4a–d). The proportion of insulin‐positive cells in the exendin‐4 or gastrin‐treated group was significantly higher than untreated control cells. Importantly, E&G co‐stimulation resulted in a significant increase in the proportion of insulin‐positive cells than monotherapy with exendin‐4 or gastrin. Western blot analysis further confirmed that insulin protein was indeed synthesized in AR42J cells cultured with exendin‐4 or gastrin and that insulin protein levels were synergistically increased by co‐stimulation with the two agents (Figure 4h).

Bottom Line: Immunohistochemistry, western blotting and RT-PCR assays were used to assess the biological effects of the two agents.   Two weeks of combination administration of exendin-4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β-cell mass in db/db mice.While gastrin had little biological effect on INS-1 β-cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin-producing cells.Co-stimulation with exendin-4 significantly enhanced gastrin-induced endocrine differentiation of AR42J precursor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Metabolism and Endocrinology.

ABSTRACT

Unlabelled: Aim/Introduction:  Preservation of β-cell mass is crucial for maintaining long-term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of β-cell mass. Here we tested the effects of gastrin and exendin-4 in type 2 diabetic animals.

Materials and methods:   The effects of exendin-4 and gastrin on β-cell function and mass were examined in 8-week-old db/db mice. INS-1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT-PCR assays were used to assess the biological effects of the two agents.

Results:   Two weeks of combination administration of exendin-4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β-cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly-shaped small islets and single insulin-positive cells. While gastrin had little biological effect on INS-1 β-cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin-producing cells. Co-stimulation with exendin-4 significantly enhanced gastrin-induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in β-cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA.

Conclusions:   These results suggest that combination treatment of db/db mice with exendin-4 and gastrin preserves β-cell mass by stimulating β-cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.00044.x, 2010).

No MeSH data available.


Related in: MedlinePlus