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Effects of High Glucose Levels and Glycated Serum on GIP Responsiveness in the Pancreatic Beta Cell Line HIT-T15.

Puddu A, Sanguineti R, Montecucco F, Viviani GL - J Diabetes Res (2015)

Bottom Line: The results showed that incubation with GS alone altered intracellular GIP signaling and decreased insulin secretion as compared to CTR.GS in combination with HG reduced the expression of GIPR and PI3K and abrogated GIP-induced AKT phosphorylation and GIP-stimulated insulin secretion.In conclusion, we showed that treatment with GS is associated with the loss of the insulinotropic effect of GIP in hyperglycemic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Genoa, Viale Benedetto XV, 16143 Genoa, Italy.

ABSTRACT
Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone produced in the gastrointestinal tract that stimulates glucose dependent insulin secretion. Impaired incretin response has been documented in diabetic patients and was mainly related to the inability of the pancreatic beta cells to secrete insulin in response to GIP. Advanced Glycation End Products (AGEs) have been shown to play an important role in pancreatic beta cell dysfunction. The aim of this study is to investigate whether the exposure to AGEs can induce GIP resistance in the pancreatic beta cell line HIT-T15. Cells were cultured for 5 days in low (CTR) or high glucose (HG) concentration in the presence of AGEs (GS) to evaluate the expression of GIP receptor (GIPR), the intracellular signaling activated by GIP, and secretion of insulin in response to GIP. The results showed that incubation with GS alone altered intracellular GIP signaling and decreased insulin secretion as compared to CTR. GS in combination with HG reduced the expression of GIPR and PI3K and abrogated GIP-induced AKT phosphorylation and GIP-stimulated insulin secretion. In conclusion, we showed that treatment with GS is associated with the loss of the insulinotropic effect of GIP in hyperglycemic conditions.

No MeSH data available.


Related in: MedlinePlus

Treatment with GS abrogates GIP-induced phosphorylation of AKT (Ser473) at both low and high glucose concentration. After 5-day treatment in the presence of standard medium (CTR) or high glucose concentration (11.1 mmol/L) (HG) in presence of glycated serum (GS), HIT-T15 cells were incubated for 2 hours in serum-free medium and then stimulated with 100 nmol/L GIP for 5 min (dark bars). (a) Representative western blot analysis. (b) Quantification of densitometries of western blot bands. Data were expressed as mean ± SE of fold induction relative to GAPDH (n = 3). ∗P < 0.05 versus absence of GIP; n. s.: nonsignificant.
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fig3: Treatment with GS abrogates GIP-induced phosphorylation of AKT (Ser473) at both low and high glucose concentration. After 5-day treatment in the presence of standard medium (CTR) or high glucose concentration (11.1 mmol/L) (HG) in presence of glycated serum (GS), HIT-T15 cells were incubated for 2 hours in serum-free medium and then stimulated with 100 nmol/L GIP for 5 min (dark bars). (a) Representative western blot analysis. (b) Quantification of densitometries of western blot bands. Data were expressed as mean ± SE of fold induction relative to GAPDH (n = 3). ∗P < 0.05 versus absence of GIP; n. s.: nonsignificant.

Mentions: Activation of GIPR signaling is coupled to increased phosphorylation of several substrates including p44/42 ERK and AKT [33]. In order to investigate whether decreased GIPR expression is associated with altered intracellular signaling, we stimulated HIT-T15 cells with 100 nmol/L GIP and then analyzed the phosphorylation of p42/44 ERK and AKT, two validated intracellular kinases downstream GIPR [33]. Treatment with GS and HG (alone or in combination) did not affect GIP-induced p42/44 ERK phosphorylation (Figures 2(a)–2(c)). On the contrary, GS and HG (alone and in combination) abrogated GIP-induced AKT activation (Figures 3(a) and 3(b)). Since PI3K is known to be the upstream activator of AKT [34], we determined whether HG or GS regulates PI3K protein expression. Treatment with HG alone or in combination with GS, but not GS alone, significantly reduced the expression of PI3K in HIT-T15 cells (Figures 4(a) and 4(b)).


Effects of High Glucose Levels and Glycated Serum on GIP Responsiveness in the Pancreatic Beta Cell Line HIT-T15.

Puddu A, Sanguineti R, Montecucco F, Viviani GL - J Diabetes Res (2015)

Treatment with GS abrogates GIP-induced phosphorylation of AKT (Ser473) at both low and high glucose concentration. After 5-day treatment in the presence of standard medium (CTR) or high glucose concentration (11.1 mmol/L) (HG) in presence of glycated serum (GS), HIT-T15 cells were incubated for 2 hours in serum-free medium and then stimulated with 100 nmol/L GIP for 5 min (dark bars). (a) Representative western blot analysis. (b) Quantification of densitometries of western blot bands. Data were expressed as mean ± SE of fold induction relative to GAPDH (n = 3). ∗P < 0.05 versus absence of GIP; n. s.: nonsignificant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499629&req=5

fig3: Treatment with GS abrogates GIP-induced phosphorylation of AKT (Ser473) at both low and high glucose concentration. After 5-day treatment in the presence of standard medium (CTR) or high glucose concentration (11.1 mmol/L) (HG) in presence of glycated serum (GS), HIT-T15 cells were incubated for 2 hours in serum-free medium and then stimulated with 100 nmol/L GIP for 5 min (dark bars). (a) Representative western blot analysis. (b) Quantification of densitometries of western blot bands. Data were expressed as mean ± SE of fold induction relative to GAPDH (n = 3). ∗P < 0.05 versus absence of GIP; n. s.: nonsignificant.
Mentions: Activation of GIPR signaling is coupled to increased phosphorylation of several substrates including p44/42 ERK and AKT [33]. In order to investigate whether decreased GIPR expression is associated with altered intracellular signaling, we stimulated HIT-T15 cells with 100 nmol/L GIP and then analyzed the phosphorylation of p42/44 ERK and AKT, two validated intracellular kinases downstream GIPR [33]. Treatment with GS and HG (alone or in combination) did not affect GIP-induced p42/44 ERK phosphorylation (Figures 2(a)–2(c)). On the contrary, GS and HG (alone and in combination) abrogated GIP-induced AKT activation (Figures 3(a) and 3(b)). Since PI3K is known to be the upstream activator of AKT [34], we determined whether HG or GS regulates PI3K protein expression. Treatment with HG alone or in combination with GS, but not GS alone, significantly reduced the expression of PI3K in HIT-T15 cells (Figures 4(a) and 4(b)).

Bottom Line: The results showed that incubation with GS alone altered intracellular GIP signaling and decreased insulin secretion as compared to CTR.GS in combination with HG reduced the expression of GIPR and PI3K and abrogated GIP-induced AKT phosphorylation and GIP-stimulated insulin secretion.In conclusion, we showed that treatment with GS is associated with the loss of the insulinotropic effect of GIP in hyperglycemic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Genoa, Viale Benedetto XV, 16143 Genoa, Italy.

ABSTRACT
Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone produced in the gastrointestinal tract that stimulates glucose dependent insulin secretion. Impaired incretin response has been documented in diabetic patients and was mainly related to the inability of the pancreatic beta cells to secrete insulin in response to GIP. Advanced Glycation End Products (AGEs) have been shown to play an important role in pancreatic beta cell dysfunction. The aim of this study is to investigate whether the exposure to AGEs can induce GIP resistance in the pancreatic beta cell line HIT-T15. Cells were cultured for 5 days in low (CTR) or high glucose (HG) concentration in the presence of AGEs (GS) to evaluate the expression of GIP receptor (GIPR), the intracellular signaling activated by GIP, and secretion of insulin in response to GIP. The results showed that incubation with GS alone altered intracellular GIP signaling and decreased insulin secretion as compared to CTR. GS in combination with HG reduced the expression of GIPR and PI3K and abrogated GIP-induced AKT phosphorylation and GIP-stimulated insulin secretion. In conclusion, we showed that treatment with GS is associated with the loss of the insulinotropic effect of GIP in hyperglycemic conditions.

No MeSH data available.


Related in: MedlinePlus