In vitro beta cell protection from STZ by PPAG.

<p>INS-1E cells were pre-treated with 30 μM PPAG for 16 hours, exposed to 1 mM streptozotocin for 1 hour and then cultured for 23 hours in medium containing 11 or 33 mM glucose with or without PPAG (n = 3). The percentage of apoptotic (A) and necrotic cells (B) was determined following staining with the nuclear dyes propidium iodide and Hoechst 33342. A minimum of 500 cells was counted for each condition. Percentage of apoptotic (A) and necrotic cells (B). *p<0.05 against control (CTRL). #p<0.05 against STZ-treated cells in 11 mM glucose. §p<0.05 as indicated.</p

In vitro beta cell protection from hydrogen peroxide by PPAG and the effect of PPAG on oxidative stress.

<p>Cell death in INS-1E cells pre-treated with 30 μM PPAG for 16 hours, exposed to 30 μM hydrogen peroxide (H₂O₂) for 30 min, and then cultured for 24 hours in control medium with or without PPAG (n = 4). The percentage of apoptotic (A) and necrotic cells (B) was determined following staining with the nuclear dyes propidium iodide and Hoechst 33342. A minimum of 500 cells was counted for each condition. Percentage of apoptotic (A) and necrotic cells (B).*p<0.05 against untreated cells. #p<0.05 against H₂O₂-treated cells. (C) INS-1E cells were pre-treated for 16 hours with 30 μM PPAG, exposed to 1 mM STZ for 1 hour, and further cultured for 2 hours in the presence of absence of PPAG (n = 3). Oxidative stress was measured by dichlorofluorescein (DCF) oxidation. *p<0.05 against untreated cells.</p

Effect of STZ and PPAG on beta cell apoptosis/proliferation and the expression of BCL2 and phospho H2AX.

<p>Shaded bars: untreated controls, CTRL (n = 5); filled bars: STZ-treated animals, STZ (n = 4); open bars: STZ-treated animals that were treated with PPAG, STZ+PPAG (n = 4). (A) Percentage of TUNEL stained cells in the insulin-positive cell population in pancreatic tissue sections. (B) Percentage of Ki67 stained cells in the insulin-positive cell population in pancreatic tissue sections. Values are mean ± SEM. Significance compared to the control was determined by one-way ANOVA with Dunnett post-test. **P<0.01; NS (not significant). For the effect of PPAG on STZ induced apoptosis a one-sample t-test was used. #P<0.05. (C) Densitometric quantification of BCL2 expression in the islets. Values are mean ± SEM. Significance compared with the control was determined using an one-way ANOVA with a Dunnett post-test. **P<0.01; NS (not significant). (D) Representative images of the BCL2 staining in pancreatic islets, 30 hours post-STZ. (E) Beta cell DNA damage/repair. Shaded bars: untreated controls, CTRL (n = 5); filled bars: STZ-treated animals, STZ (n = 4 for 30 hours and n = 3 for 11 days); open bars: STZ-treated animals that were treated with PPAG, STZ+PPAG (n = 4 for 30 hours and n = 4 for 11 days). No positive cells were detected in control animals. Results from STZ and STZ+PPAG were not statistically different (p≥0.05). **p<0.01 between control group and STZ.</p

Role of the Saturated Nonesterified Fatty Acid Palmitate in Beta Cell Dysfunction

Sustained elevated levels of saturated free fatty acids, such as palmitate, contribute to beta cell dysfunction, a phenomenon aggravated by high glucose levels. The aim of this study was to investigate the mechanisms of palmitate-induced beta cell dysfunction and death, combined or not with high glucose. Protein profiling of INS-1E cells, exposed to 0.5 mmol/L palmitate and combined or not with 25 mmol/L glucose, for 24 h was done by 2D-DIGE, both on full cell lysate and on an enriched endoplasmic reticulum (ER) fraction. Eighty-three differentially expressed proteins (<i>P</i> < 0.05) were identified by MALDI-TOF/TOF mass spectrometry and proteomic results were confirmed by functional assays. 2D-DIGE analysis of whole cell lysates and ER enriched samples revealed a high number of proteins compared to previous reports. Palmitate induced beta cell dysfunction and death via ER stress, hampered insulin maturation, generation of harmful metabolites during triglycerides synthesis and altered intracellular trafficking. In combination with high glucose, palmitate induced increased shunting of excess glucose, increased mitochondrial reactive oxygen species production and an elevation in many transcription-related proteins. This study contributes to a better understanding and revealed novel mechanisms of palmitate-induced beta cell dysfunction and death and may provide new targets for drug discovery

Role of the Saturated Nonesterified Fatty Acid Palmitate in Beta Cell Dysfunction

Sustained elevated levels of saturated free fatty acids, such as palmitate, contribute to beta cell dysfunction, a phenomenon aggravated by high glucose levels. The aim of this study was to investigate the mechanisms of palmitate-induced beta cell dysfunction and death, combined or not with high glucose. Protein profiling of INS-1E cells, exposed to 0.5 mmol/L palmitate and combined or not with 25 mmol/L glucose, for 24 h was done by 2D-DIGE, both on full cell lysate and on an enriched endoplasmic reticulum (ER) fraction. Eighty-three differentially expressed proteins (<i>P</i> < 0.05) were identified by MALDI-TOF/TOF mass spectrometry and proteomic results were confirmed by functional assays. 2D-DIGE analysis of whole cell lysates and ER enriched samples revealed a high number of proteins compared to previous reports. Palmitate induced beta cell dysfunction and death via ER stress, hampered insulin maturation, generation of harmful metabolites during triglycerides synthesis and altered intracellular trafficking. In combination with high glucose, palmitate induced increased shunting of excess glucose, increased mitochondrial reactive oxygen species production and an elevation in many transcription-related proteins. This study contributes to a better understanding and revealed novel mechanisms of palmitate-induced beta cell dysfunction and death and may provide new targets for drug discovery

Effect of PPAG on lipotoxic cell death in human islet cells.

<p>Human islets were treated with 0.5 mM palmitate and 30 μM PPAG or vehicle for 3 days (n = 4). The percentage of apoptotic (A) and necrotic cells (B) was determined following staining with the nuclear dyes propidium iodide and Hoechst 33342. A minimum of 500 cells was counted for each condition. *p<0.05 against untreated cells, #p< 0.05 as indicated.</p

Effect of STZ and PPAG on blood glucose levels and beta cell mass in vivo.

<p>Shaded bars: untreated controls, CTRL (n = 10); filled bars: STZ-treated animals, STZ (n = 7); open bars: STZ-treated animals that were treated with PPAG, STZ+PPAG (n = 8). (A) Non-fasting blood glucose levels immediately before and 30 hours after a single injection of STZ. (B) Daily follow up of non-fasting blood glucose from immediately before a single injection of STZ until 11 days. Values are mean ± SEM. ANOVA with Bonferroni post test was used to determine the significance between groups on each day. **p<0.01; ***p<0.001 between control and STZ group. #p<0.05; ## p<0.01; ###p< 0.001 between control and STZ + PPAG group. § p<0.05; §§ p<0.01; §§§ p< 0.001 between STZ and STZ + PPAG group. (C) Percentage of beta cell mass normalized to the control 30 hours (C) or 11 days (D) after STZ injection. Data shown are means ± SEM. **p<0.01; #p<0.05; NS: not significant.</p

Expression of caspases and BCL2.

<p>Western blot and densitometry for BCL2 and cleaved caspase 9 and 3 in INS-1E cells pre-treated with 30 μM PPAG for 16 hours, exposed to 1 mM STZ for 1 hour and then cultured for 7 hours in medium containing 11 or 33 mM glucose with or without PPAG (n = 3).*p<0.05 against control (CTRL). §p<0.05 as indicated.</p

Role of the Saturated Nonesterified Fatty Acid Palmitate in Beta Cell Dysfunction

Sustained elevated levels of saturated free fatty acids, such as palmitate, contribute to beta cell dysfunction, a phenomenon aggravated by high glucose levels. The aim of this study was to investigate the mechanisms of palmitate-induced beta cell dysfunction and death, combined or not with high glucose. Protein profiling of INS-1E cells, exposed to 0.5 mmol/L palmitate and combined or not with 25 mmol/L glucose, for 24 h was done by 2D-DIGE, both on full cell lysate and on an enriched endoplasmic reticulum (ER) fraction. Eighty-three differentially expressed proteins (<i>P</i> < 0.05) were identified by MALDI-TOF/TOF mass spectrometry and proteomic results were confirmed by functional assays. 2D-DIGE analysis of whole cell lysates and ER enriched samples revealed a high number of proteins compared to previous reports. Palmitate induced beta cell dysfunction and death via ER stress, hampered insulin maturation, generation of harmful metabolites during triglycerides synthesis and altered intracellular trafficking. In combination with high glucose, palmitate induced increased shunting of excess glucose, increased mitochondrial reactive oxygen species production and an elevation in many transcription-related proteins. This study contributes to a better understanding and revealed novel mechanisms of palmitate-induced beta cell dysfunction and death and may provide new targets for drug discovery

Role of the Saturated Nonesterified Fatty Acid Palmitate in Beta Cell Dysfunction

Sustained elevated levels of saturated free fatty acids, such as palmitate, contribute to beta cell dysfunction, a phenomenon aggravated by high glucose levels. The aim of this study was to investigate the mechanisms of palmitate-induced beta cell dysfunction and death, combined or not with high glucose. Protein profiling of INS-1E cells, exposed to 0.5 mmol/L palmitate and combined or not with 25 mmol/L glucose, for 24 h was done by 2D-DIGE, both on full cell lysate and on an enriched endoplasmic reticulum (ER) fraction. Eighty-three differentially expressed proteins (<i>P</i> < 0.05) were identified by MALDI-TOF/TOF mass spectrometry and proteomic results were confirmed by functional assays. 2D-DIGE analysis of whole cell lysates and ER enriched samples revealed a high number of proteins compared to previous reports. Palmitate induced beta cell dysfunction and death via ER stress, hampered insulin maturation, generation of harmful metabolites during triglycerides synthesis and altered intracellular trafficking. In combination with high glucose, palmitate induced increased shunting of excess glucose, increased mitochondrial reactive oxygen species production and an elevation in many transcription-related proteins. This study contributes to a better understanding and revealed novel mechanisms of palmitate-induced beta cell dysfunction and death and may provide new targets for drug discovery