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The diabetes-linked transcription factor PAX4 promotes {beta}-cell proliferation and survival in rat and human islets.

Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB, Gauthier BR - J. Cell Biol. (2004)

Bottom Line: Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively.Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient.We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland. Thierry.brun@medecine.unige.ch

ABSTRACT
The mechanism by which the beta-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

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Total cellular ATP and mitochondrial calcium levels are increased in AdCMVPax4IRESGFP-infected islets. (A) Total cellular ATP levels were measured in islets overexpressing either β-galactosidase or PAX4 (2.4 × 107 pfu/ml, 50% of cell infected) and maintained in 1 mM glucose for 10 min. Results represent the means ± SEM. **, P < 0.01. (B) Cytosolic ATP production in response to 2.5 or 16.5 mM glucose was determined over a period of 20 min using the ATP-sensitive bioluminescence probe luciferase (3.6 × 107 pfu/ml). Glucose and azide were added at indicated times (arrows). Results are the mean ± SEM of at least five experiments performed in duplicates (*, P < 0.05). (C) Mitochondrial calcium was monitored in β-galactosidase or PAX4 overexpressing islets using β-cell–specific/mitochondrial-targeted aequorin as described in Materials and methods. After the establishment of baseline luminescence (30 min; LacZ = 210 ± 49 nM and Pax4 = 387 ± 46 nM, left), islets were superfused for 5 min in basal conditions (0 glucose) before stimulation with glucose (16.7 mM), and then KCl (60 mM) for 5 min intervals each, as shown (middle). The induced increases in [Ca2+]m were evaluated on the basis of the AUP and a presented on the right. Each value represents the mean ± SEM of a minimum of six separate experiments. *, P < 0.05. (D) Proposed model of Pax4-induced β-cell proliferation. Mitogens activate Pax4, which will stimulate c-myc and Bcl-xL gene transcription. c-Myc will promote Id2 gene expression and activate the cell cycle replication program. Bcl-xL increased expression will promote survival by preventing mitochondria from initiating the apoptotic program. However, cells become refractory to glucose-evoked insulin secretion due to altered ATP production and calcium handling.
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fig6: Total cellular ATP and mitochondrial calcium levels are increased in AdCMVPax4IRESGFP-infected islets. (A) Total cellular ATP levels were measured in islets overexpressing either β-galactosidase or PAX4 (2.4 × 107 pfu/ml, 50% of cell infected) and maintained in 1 mM glucose for 10 min. Results represent the means ± SEM. **, P < 0.01. (B) Cytosolic ATP production in response to 2.5 or 16.5 mM glucose was determined over a period of 20 min using the ATP-sensitive bioluminescence probe luciferase (3.6 × 107 pfu/ml). Glucose and azide were added at indicated times (arrows). Results are the mean ± SEM of at least five experiments performed in duplicates (*, P < 0.05). (C) Mitochondrial calcium was monitored in β-galactosidase or PAX4 overexpressing islets using β-cell–specific/mitochondrial-targeted aequorin as described in Materials and methods. After the establishment of baseline luminescence (30 min; LacZ = 210 ± 49 nM and Pax4 = 387 ± 46 nM, left), islets were superfused for 5 min in basal conditions (0 glucose) before stimulation with glucose (16.7 mM), and then KCl (60 mM) for 5 min intervals each, as shown (middle). The induced increases in [Ca2+]m were evaluated on the basis of the AUP and a presented on the right. Each value represents the mean ± SEM of a minimum of six separate experiments. *, P < 0.05. (D) Proposed model of Pax4-induced β-cell proliferation. Mitogens activate Pax4, which will stimulate c-myc and Bcl-xL gene transcription. c-Myc will promote Id2 gene expression and activate the cell cycle replication program. Bcl-xL increased expression will promote survival by preventing mitochondria from initiating the apoptotic program. However, cells become refractory to glucose-evoked insulin secretion due to altered ATP production and calcium handling.

Mentions: Although other antiapoptotic genes may be implicated in the protection of c-myc–induced cell death, we pursued the potential protective function of Bcl-xL in view of its link with c-myc in β-cell survival and proliferation (Pelengaris et al., 2002). Small increases in Bcl-xL, similar to those observed in our work, were shown to protect β-cells against thapsigargin-induced apoptosis in a transgenic mouse model. Increased levels of this mitochondrially targeted protein were also found to impair insulin secretion (Zhou et al., 2000). Consistent with these studies, we found that glucose-stimulated insulin exocytosis was attenuated by 50% in Pax4-overexpressing islets 48 h after infection. β-Galactosidase–expressing islets and noninfected controls exhibited an expected threefold increase in hormone release (Fig. 5 A). However, inhibition was transient as glucose-induced insulin secretion was restored 6 d after infection (unpublished data). Inclusion of 1 μM forskolin/100 μM IBMX, which modulates the effect of glucose on secretion by raising cAMP levels, restored glucose-induced insulin exocytosis, indicating that events downstream of plasma membrane depolarization are functional in Pax4-expressing cells. To evaluate whether or not Pax4-induced Bcl-xL expression curtails the metabolism–secretion coupling cascade, glucose metabolism as well as ATP levels and mitochondrial calcium concentrations ([Ca2+]m) were measured in transduced islets. The rate of glucose oxidation was estimated by measuring the conversion of D-[14C(U)] to 14CO2 and found to be equally efficient in both control and infected islets (Fig. 5 B). However, total cellular ATP levels were fourfold higher in islets expressing Pax4 as compared with control LacZ islets (Fig. 6 A). Cellular ATP levels largely reflect sequestered pools in organelles, in particular in the mitochondria (Detimary et al., 1995). These results prompted us to investigate whether or not glucose was able to raise cytosolic ATP levels in Pax4-overexpressing islets, which are essential in the coupling of metabolism to insulin secretion (Gauthier et al., 2004). Addition of 16.5 mM glucose to control/LacZ islets resulted in a 23% increase of cytosolic ATP, which was sustained until the injection of azide, a compound that dissipates the mitochondrial membrane potential and thus interrupts ATP formation (Fig. 6 B). Cytosolic ATP from islets maintained in 2.5 mM glucose gradually decreased to levels 80% of those at time of glucose injection consistent with low sustained energy consumption. Unexpectedly, basal cytosolic ATP in AdCMVPax4IRESGFP-infected islets was 30% of that measured in control islets, and a small nonsignificant increase in production was detected after exposure to 16.5 mM glucose (Fig. 6 B). Changes in cytosolic calcium are relayed to the mitochondria (Kennedy et al., 1996; Ishihara et al., 2003). Resting [Ca2+]m was elevated in β-cells of Pax4-transduced islets, nearly twofold higher than controls (Fig. 6 C). High concentrations of extracellular potassium trigger calcium influx across the plasma membrane independently of ATP production and KATP channel closure. The potassium-induced rise in [Ca2+]m was normal in transduced islets, as assessed by the total increase in [Ca2+]m (area under peak [AUP]). However, the glucose-induced increase in [Ca2+]m (AUP) was attenuated by 40 ± 5% in Pax4-expressing islets. Together, these results indicate that increased Pax4 expression provokes alterations in both mitochondrial calcium levels and ATP synthesis, which may underlie the blunted glucose-induced insulin secretion (Fig. 6 D).


The diabetes-linked transcription factor PAX4 promotes {beta}-cell proliferation and survival in rat and human islets.

Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB, Gauthier BR - J. Cell Biol. (2004)

Total cellular ATP and mitochondrial calcium levels are increased in AdCMVPax4IRESGFP-infected islets. (A) Total cellular ATP levels were measured in islets overexpressing either β-galactosidase or PAX4 (2.4 × 107 pfu/ml, 50% of cell infected) and maintained in 1 mM glucose for 10 min. Results represent the means ± SEM. **, P < 0.01. (B) Cytosolic ATP production in response to 2.5 or 16.5 mM glucose was determined over a period of 20 min using the ATP-sensitive bioluminescence probe luciferase (3.6 × 107 pfu/ml). Glucose and azide were added at indicated times (arrows). Results are the mean ± SEM of at least five experiments performed in duplicates (*, P < 0.05). (C) Mitochondrial calcium was monitored in β-galactosidase or PAX4 overexpressing islets using β-cell–specific/mitochondrial-targeted aequorin as described in Materials and methods. After the establishment of baseline luminescence (30 min; LacZ = 210 ± 49 nM and Pax4 = 387 ± 46 nM, left), islets were superfused for 5 min in basal conditions (0 glucose) before stimulation with glucose (16.7 mM), and then KCl (60 mM) for 5 min intervals each, as shown (middle). The induced increases in [Ca2+]m were evaluated on the basis of the AUP and a presented on the right. Each value represents the mean ± SEM of a minimum of six separate experiments. *, P < 0.05. (D) Proposed model of Pax4-induced β-cell proliferation. Mitogens activate Pax4, which will stimulate c-myc and Bcl-xL gene transcription. c-Myc will promote Id2 gene expression and activate the cell cycle replication program. Bcl-xL increased expression will promote survival by preventing mitochondria from initiating the apoptotic program. However, cells become refractory to glucose-evoked insulin secretion due to altered ATP production and calcium handling.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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fig6: Total cellular ATP and mitochondrial calcium levels are increased in AdCMVPax4IRESGFP-infected islets. (A) Total cellular ATP levels were measured in islets overexpressing either β-galactosidase or PAX4 (2.4 × 107 pfu/ml, 50% of cell infected) and maintained in 1 mM glucose for 10 min. Results represent the means ± SEM. **, P < 0.01. (B) Cytosolic ATP production in response to 2.5 or 16.5 mM glucose was determined over a period of 20 min using the ATP-sensitive bioluminescence probe luciferase (3.6 × 107 pfu/ml). Glucose and azide were added at indicated times (arrows). Results are the mean ± SEM of at least five experiments performed in duplicates (*, P < 0.05). (C) Mitochondrial calcium was monitored in β-galactosidase or PAX4 overexpressing islets using β-cell–specific/mitochondrial-targeted aequorin as described in Materials and methods. After the establishment of baseline luminescence (30 min; LacZ = 210 ± 49 nM and Pax4 = 387 ± 46 nM, left), islets were superfused for 5 min in basal conditions (0 glucose) before stimulation with glucose (16.7 mM), and then KCl (60 mM) for 5 min intervals each, as shown (middle). The induced increases in [Ca2+]m were evaluated on the basis of the AUP and a presented on the right. Each value represents the mean ± SEM of a minimum of six separate experiments. *, P < 0.05. (D) Proposed model of Pax4-induced β-cell proliferation. Mitogens activate Pax4, which will stimulate c-myc and Bcl-xL gene transcription. c-Myc will promote Id2 gene expression and activate the cell cycle replication program. Bcl-xL increased expression will promote survival by preventing mitochondria from initiating the apoptotic program. However, cells become refractory to glucose-evoked insulin secretion due to altered ATP production and calcium handling.
Mentions: Although other antiapoptotic genes may be implicated in the protection of c-myc–induced cell death, we pursued the potential protective function of Bcl-xL in view of its link with c-myc in β-cell survival and proliferation (Pelengaris et al., 2002). Small increases in Bcl-xL, similar to those observed in our work, were shown to protect β-cells against thapsigargin-induced apoptosis in a transgenic mouse model. Increased levels of this mitochondrially targeted protein were also found to impair insulin secretion (Zhou et al., 2000). Consistent with these studies, we found that glucose-stimulated insulin exocytosis was attenuated by 50% in Pax4-overexpressing islets 48 h after infection. β-Galactosidase–expressing islets and noninfected controls exhibited an expected threefold increase in hormone release (Fig. 5 A). However, inhibition was transient as glucose-induced insulin secretion was restored 6 d after infection (unpublished data). Inclusion of 1 μM forskolin/100 μM IBMX, which modulates the effect of glucose on secretion by raising cAMP levels, restored glucose-induced insulin exocytosis, indicating that events downstream of plasma membrane depolarization are functional in Pax4-expressing cells. To evaluate whether or not Pax4-induced Bcl-xL expression curtails the metabolism–secretion coupling cascade, glucose metabolism as well as ATP levels and mitochondrial calcium concentrations ([Ca2+]m) were measured in transduced islets. The rate of glucose oxidation was estimated by measuring the conversion of D-[14C(U)] to 14CO2 and found to be equally efficient in both control and infected islets (Fig. 5 B). However, total cellular ATP levels were fourfold higher in islets expressing Pax4 as compared with control LacZ islets (Fig. 6 A). Cellular ATP levels largely reflect sequestered pools in organelles, in particular in the mitochondria (Detimary et al., 1995). These results prompted us to investigate whether or not glucose was able to raise cytosolic ATP levels in Pax4-overexpressing islets, which are essential in the coupling of metabolism to insulin secretion (Gauthier et al., 2004). Addition of 16.5 mM glucose to control/LacZ islets resulted in a 23% increase of cytosolic ATP, which was sustained until the injection of azide, a compound that dissipates the mitochondrial membrane potential and thus interrupts ATP formation (Fig. 6 B). Cytosolic ATP from islets maintained in 2.5 mM glucose gradually decreased to levels 80% of those at time of glucose injection consistent with low sustained energy consumption. Unexpectedly, basal cytosolic ATP in AdCMVPax4IRESGFP-infected islets was 30% of that measured in control islets, and a small nonsignificant increase in production was detected after exposure to 16.5 mM glucose (Fig. 6 B). Changes in cytosolic calcium are relayed to the mitochondria (Kennedy et al., 1996; Ishihara et al., 2003). Resting [Ca2+]m was elevated in β-cells of Pax4-transduced islets, nearly twofold higher than controls (Fig. 6 C). High concentrations of extracellular potassium trigger calcium influx across the plasma membrane independently of ATP production and KATP channel closure. The potassium-induced rise in [Ca2+]m was normal in transduced islets, as assessed by the total increase in [Ca2+]m (area under peak [AUP]). However, the glucose-induced increase in [Ca2+]m (AUP) was attenuated by 40 ± 5% in Pax4-expressing islets. Together, these results indicate that increased Pax4 expression provokes alterations in both mitochondrial calcium levels and ATP synthesis, which may underlie the blunted glucose-induced insulin secretion (Fig. 6 D).

Bottom Line: Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively.Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient.We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland. Thierry.brun@medecine.unige.ch

ABSTRACT
The mechanism by which the beta-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in beta-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in beta-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.

Show MeSH
Related in: MedlinePlus