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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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Effects of Pax4 overexpression on insulin secretion and glucose oxidation in isolated rat islets. (A) Glucose-induced insulin secretion was inhibited by AdCMVPax4IRESGFP. 2 d after infection, islet hormone secretion was assayed as described in Materials and methods. Data are expressed as the mean ± SEM of four independent experiments. **, P < 0.01. (B) 2 d after infection with 2.4 × 107 pfu/ml of indicated adenoviruses, islet CO2 generation was measured in the presence of 2.5 or 16.7 mM glucose to assess glucose oxidation rate as described in the experimental procedures. Data represent the mean ± SEM of five independent experiments.
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fig5: Effects of Pax4 overexpression on insulin secretion and glucose oxidation in isolated rat islets. (A) Glucose-induced insulin secretion was inhibited by AdCMVPax4IRESGFP. 2 d after infection, islet hormone secretion was assayed as described in Materials and methods. Data are expressed as the mean ± SEM of four independent experiments. **, P < 0.01. (B) 2 d after infection with 2.4 × 107 pfu/ml of indicated adenoviruses, islet CO2 generation was measured in the presence of 2.5 or 16.7 mM glucose to assess glucose oxidation rate as described in the experimental procedures. Data represent the mean ± SEM of five independent experiments.

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)

Effects of Pax4 overexpression on insulin secretion and glucose oxidation in isolated rat islets. (A) Glucose-induced insulin secretion was inhibited by AdCMVPax4IRESGFP. 2 d after infection, islet hormone secretion was assayed as described in Materials and methods. Data are expressed as the mean ± SEM of four independent experiments. **, P < 0.01. (B) 2 d after infection with 2.4 × 107 pfu/ml of indicated adenoviruses, islet CO2 generation was measured in the presence of 2.5 or 16.7 mM glucose to assess glucose oxidation rate as described in the experimental procedures. Data represent the mean ± SEM of five independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172618&req=5

fig5: Effects of Pax4 overexpression on insulin secretion and glucose oxidation in isolated rat islets. (A) Glucose-induced insulin secretion was inhibited by AdCMVPax4IRESGFP. 2 d after infection, islet hormone secretion was assayed as described in Materials and methods. Data are expressed as the mean ± SEM of four independent experiments. **, P < 0.01. (B) 2 d after infection with 2.4 × 107 pfu/ml of indicated adenoviruses, islet CO2 generation was measured in the presence of 2.5 or 16.7 mM glucose to assess glucose oxidation rate as described in the experimental procedures. Data represent the mean ± SEM of five independent experiments.
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