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Squamosamide Derivative FLZ Protects Pancreatic β-Cells from Glucotoxicity by Stimulating Akt-FOXO1 Pathway.

Kong X, Zhang L, Hua X, Ma X - J Diabetes Res (2015)

Bottom Line: On the contrary, FLZ increased pancreatic and duodenal homeobox-1 expression and its nuclear localization, an effect mediated by increased p-Akt.Consistently, Akt selective inhibitor MK-2206 completely abolished antiglucotoxicity effect of FLZ.Taken together, our results suggest that FLZ could be a potential therapeutic agent to treat the hyperglycemia-induced β-cell failure.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, Shenzhen University, Shenzhen 518060, China.

ABSTRACT
Chronic hyperglycemia increases apoptosis and reduces glucose-stimulated insulin secretion. Although protective agents have been searched extensively, none has been found so far. Here we tested FLZ, a synthetic derivative of squamosamide from a Chinese herb, as a potential candidate for antiglucotoxicity in INS-1E cells and mouse islets. Chronic culture of β-cells in 30 mM glucose caused progressive reduction of cell viability, accompanied with increased apoptosis and reduced insulin secretion. These effects on apoptosis and insulin were reversed by FLZ in a dose-dependent manner. FLZ treatment also increased forkhead box O1 protein phosphorylation and reduced its nuclear location. On the contrary, FLZ increased pancreatic and duodenal homeobox-1 expression and its nuclear localization, an effect mediated by increased p-Akt. Consistently, Akt selective inhibitor MK-2206 completely abolished antiglucotoxicity effect of FLZ. Furthermore, FLZ treatment increased cytosolic ATP/ADP ratio. Taken together, our results suggest that FLZ could be a potential therapeutic agent to treat the hyperglycemia-induced β-cell failure.

No MeSH data available.


Related in: MedlinePlus

Treatment with FLZ prevents glucotoxicity in INS-1E cells. (a) INS-1E cells were cultured at G11.1 and G30 for 3–5 days and cell viability was assessed with MTT assay. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3–6 independent experiments. ∗∗p < 0.01 versus the value at G11.1. (b) Cell viability was assessed in INS-1E cells being cultured at G11.1 and G30 for 4 days. Cells were treated without or with varying levels of FLZ. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3 independent experiments. ∗∗p < 0.01 versus the value at G11.1; #p < 0.05, ##p < 0.01 versus the value at G30 without FLZ. (c) Apoptosis assessed by HO/PI staining in INS-1E cells cultured at G11.1 and G30 for 4 days. Cells were treated without or with 10 μM FLZ. Arrows indicate HO-stained cells with apoptotic bodies (upper panel). Mean values ± S.E.M. of 3 independent experiments (low panel). ∗∗p < 0.01 versus the value at G11.1; ##p < 0.01 versus the value at G30 without FLZ. (d) INS-1E cells were cultured at G11.1 or G30 for 4 days. Cells were treated without or with 10 μM FLZ. Expression of total and cleaved caspase 3 protein was determined by western blot. β-actin was used as an internal control (upper panel). Intensities of total and cleaved caspase 3 protein expression were quantified, normalized against the level of β-actin, and expressed as fold of protein abundance in INS-1E cells at G11.1 (low panel). Data are means ± S.E.M. of 3 separate experiments. ∗p < 0.05, ∗∗p < 0.01 versus the value at G11.1; #p < 0.05 versus the value at G30 without FLZ.
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fig1: Treatment with FLZ prevents glucotoxicity in INS-1E cells. (a) INS-1E cells were cultured at G11.1 and G30 for 3–5 days and cell viability was assessed with MTT assay. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3–6 independent experiments. ∗∗p < 0.01 versus the value at G11.1. (b) Cell viability was assessed in INS-1E cells being cultured at G11.1 and G30 for 4 days. Cells were treated without or with varying levels of FLZ. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3 independent experiments. ∗∗p < 0.01 versus the value at G11.1; #p < 0.05, ##p < 0.01 versus the value at G30 without FLZ. (c) Apoptosis assessed by HO/PI staining in INS-1E cells cultured at G11.1 and G30 for 4 days. Cells were treated without or with 10 μM FLZ. Arrows indicate HO-stained cells with apoptotic bodies (upper panel). Mean values ± S.E.M. of 3 independent experiments (low panel). ∗∗p < 0.01 versus the value at G11.1; ##p < 0.01 versus the value at G30 without FLZ. (d) INS-1E cells were cultured at G11.1 or G30 for 4 days. Cells were treated without or with 10 μM FLZ. Expression of total and cleaved caspase 3 protein was determined by western blot. β-actin was used as an internal control (upper panel). Intensities of total and cleaved caspase 3 protein expression were quantified, normalized against the level of β-actin, and expressed as fold of protein abundance in INS-1E cells at G11.1 (low panel). Data are means ± S.E.M. of 3 separate experiments. ∗p < 0.05, ∗∗p < 0.01 versus the value at G11.1; #p < 0.05 versus the value at G30 without FLZ.

Mentions: Insulin-secreting INS-1E cells and mouse islets were cultured in 30 mM glucose (G30) for 3 to 5 days, a commonly used model for glucotoxicity [9, 10]. As shown in Figure 1(a), chronic culture of INS-1E cells in G30 resulted in marked decrease in cell viability as assayed by MTT and the value reduced by ~25% (3 days), ~52% (4 days), and ~75% (5 days) compared to that cultured in 11.1 mM glucose (G11.1), as reported previously [11]. Treatment with increasing concentrations of FLZ (0.1, 1, 10, and 20 μM) reversed the effect of 30 mM glucose in a dose-dependent manner (Figure 1(b)). A concentration of 10 μM of FLZ was used in most of experiments of this study, as this concentration corresponds to a maximal effective concentration reported in other studies [6]. The effect of FLZ on β-cell glucotoxicity was also investigated by measuring apoptotic cells as determined by HO/PI staining (Figure 1(c)). Chronic culture at G30 led to an increase in the percentages of apoptotic cells from 4 ± 0.1% to 37 ± 6% at G11.1, whereas FLZ treatment reduced the level of apoptosis to 10 ± 1% (p < 0.01) (Figure 1(c)). Notably, addition of FLZ essentially blocked glucotoxicity-induced increase of cleaved caspase 3 (Figure 1(d)), an activated proapoptotic protein. Thus, FLZ protects β-cells from glucotoxicity-induced cell death at least partly by preventing activation of caspase 3.


Squamosamide Derivative FLZ Protects Pancreatic β-Cells from Glucotoxicity by Stimulating Akt-FOXO1 Pathway.

Kong X, Zhang L, Hua X, Ma X - J Diabetes Res (2015)

Treatment with FLZ prevents glucotoxicity in INS-1E cells. (a) INS-1E cells were cultured at G11.1 and G30 for 3–5 days and cell viability was assessed with MTT assay. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3–6 independent experiments. ∗∗p < 0.01 versus the value at G11.1. (b) Cell viability was assessed in INS-1E cells being cultured at G11.1 and G30 for 4 days. Cells were treated without or with varying levels of FLZ. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3 independent experiments. ∗∗p < 0.01 versus the value at G11.1; #p < 0.05, ##p < 0.01 versus the value at G30 without FLZ. (c) Apoptosis assessed by HO/PI staining in INS-1E cells cultured at G11.1 and G30 for 4 days. Cells were treated without or with 10 μM FLZ. Arrows indicate HO-stained cells with apoptotic bodies (upper panel). Mean values ± S.E.M. of 3 independent experiments (low panel). ∗∗p < 0.01 versus the value at G11.1; ##p < 0.01 versus the value at G30 without FLZ. (d) INS-1E cells were cultured at G11.1 or G30 for 4 days. Cells were treated without or with 10 μM FLZ. Expression of total and cleaved caspase 3 protein was determined by western blot. β-actin was used as an internal control (upper panel). Intensities of total and cleaved caspase 3 protein expression were quantified, normalized against the level of β-actin, and expressed as fold of protein abundance in INS-1E cells at G11.1 (low panel). Data are means ± S.E.M. of 3 separate experiments. ∗p < 0.05, ∗∗p < 0.01 versus the value at G11.1; #p < 0.05 versus the value at G30 without FLZ.
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fig1: Treatment with FLZ prevents glucotoxicity in INS-1E cells. (a) INS-1E cells were cultured at G11.1 and G30 for 3–5 days and cell viability was assessed with MTT assay. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3–6 independent experiments. ∗∗p < 0.01 versus the value at G11.1. (b) Cell viability was assessed in INS-1E cells being cultured at G11.1 and G30 for 4 days. Cells were treated without or with varying levels of FLZ. Values were expressed as percentage of cell viability at G11.1. Data are means ± S.E.M. of 3 independent experiments. ∗∗p < 0.01 versus the value at G11.1; #p < 0.05, ##p < 0.01 versus the value at G30 without FLZ. (c) Apoptosis assessed by HO/PI staining in INS-1E cells cultured at G11.1 and G30 for 4 days. Cells were treated without or with 10 μM FLZ. Arrows indicate HO-stained cells with apoptotic bodies (upper panel). Mean values ± S.E.M. of 3 independent experiments (low panel). ∗∗p < 0.01 versus the value at G11.1; ##p < 0.01 versus the value at G30 without FLZ. (d) INS-1E cells were cultured at G11.1 or G30 for 4 days. Cells were treated without or with 10 μM FLZ. Expression of total and cleaved caspase 3 protein was determined by western blot. β-actin was used as an internal control (upper panel). Intensities of total and cleaved caspase 3 protein expression were quantified, normalized against the level of β-actin, and expressed as fold of protein abundance in INS-1E cells at G11.1 (low panel). Data are means ± S.E.M. of 3 separate experiments. ∗p < 0.05, ∗∗p < 0.01 versus the value at G11.1; #p < 0.05 versus the value at G30 without FLZ.
Mentions: Insulin-secreting INS-1E cells and mouse islets were cultured in 30 mM glucose (G30) for 3 to 5 days, a commonly used model for glucotoxicity [9, 10]. As shown in Figure 1(a), chronic culture of INS-1E cells in G30 resulted in marked decrease in cell viability as assayed by MTT and the value reduced by ~25% (3 days), ~52% (4 days), and ~75% (5 days) compared to that cultured in 11.1 mM glucose (G11.1), as reported previously [11]. Treatment with increasing concentrations of FLZ (0.1, 1, 10, and 20 μM) reversed the effect of 30 mM glucose in a dose-dependent manner (Figure 1(b)). A concentration of 10 μM of FLZ was used in most of experiments of this study, as this concentration corresponds to a maximal effective concentration reported in other studies [6]. The effect of FLZ on β-cell glucotoxicity was also investigated by measuring apoptotic cells as determined by HO/PI staining (Figure 1(c)). Chronic culture at G30 led to an increase in the percentages of apoptotic cells from 4 ± 0.1% to 37 ± 6% at G11.1, whereas FLZ treatment reduced the level of apoptosis to 10 ± 1% (p < 0.01) (Figure 1(c)). Notably, addition of FLZ essentially blocked glucotoxicity-induced increase of cleaved caspase 3 (Figure 1(d)), an activated proapoptotic protein. Thus, FLZ protects β-cells from glucotoxicity-induced cell death at least partly by preventing activation of caspase 3.

Bottom Line: On the contrary, FLZ increased pancreatic and duodenal homeobox-1 expression and its nuclear localization, an effect mediated by increased p-Akt.Consistently, Akt selective inhibitor MK-2206 completely abolished antiglucotoxicity effect of FLZ.Taken together, our results suggest that FLZ could be a potential therapeutic agent to treat the hyperglycemia-induced β-cell failure.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, Shenzhen University, Shenzhen 518060, China.

ABSTRACT
Chronic hyperglycemia increases apoptosis and reduces glucose-stimulated insulin secretion. Although protective agents have been searched extensively, none has been found so far. Here we tested FLZ, a synthetic derivative of squamosamide from a Chinese herb, as a potential candidate for antiglucotoxicity in INS-1E cells and mouse islets. Chronic culture of β-cells in 30 mM glucose caused progressive reduction of cell viability, accompanied with increased apoptosis and reduced insulin secretion. These effects on apoptosis and insulin were reversed by FLZ in a dose-dependent manner. FLZ treatment also increased forkhead box O1 protein phosphorylation and reduced its nuclear location. On the contrary, FLZ increased pancreatic and duodenal homeobox-1 expression and its nuclear localization, an effect mediated by increased p-Akt. Consistently, Akt selective inhibitor MK-2206 completely abolished antiglucotoxicity effect of FLZ. Furthermore, FLZ treatment increased cytosolic ATP/ADP ratio. Taken together, our results suggest that FLZ could be a potential therapeutic agent to treat the hyperglycemia-induced β-cell failure.

No MeSH data available.


Related in: MedlinePlus