Increased Expression of Antioxidant and Antiapoptotic Genes in Islets That May Contribute to β-Cell Survival During Chronic Hyperglycemia
Article Figures & Tables
Figures
- FIG. 1.
Validation of multiplex PCR amplification of glutathione peroxidase (•) using TBP (○) as an internal control. A: cDNA corresponding to 20 ng of starting RNA was amplified by an increasing number of PCR cycles and products separated on a 6% polyacrylamide gel. Band intensity was plotted as arbitrary logarithmic units to verify that all products were within the exponential phase of amplification. B: Increasing amounts of cDNA (2.5–80 ng RNA equivalent) were amplified with 27 cycles of PCR to verify that each product increased linearly with the amount of starting material. GPX, glutathione peroxidase.
- FIG. 2.
Comparison of stress gene mRNA levels in islets of sham and hyperglycemic 90% pancreatectomized (Px) rats. mRNA levels were compared by semiquantitative multiplex RT-PCR. After normalization of the specific gene to an internal control gene (cyclophilin, TBP, or α-tubulin), mRNA levels in Px islets are expressed as a percent of sham. Values are means ± SE determined from nine sham and seven Px rats. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham for each gene. Average blood glucose: sham 80 ± 2 mg/dl, Px 232 ± 14 mg/dl; P < 0.001. S, sham.
- FIG. 3.
Changes in stress gene mRNA levels in rats with different degrees of hyperglycemia 4 weeks after Px. A: Changes in blood glucose of rats in which the proportion of the pancreas removed was varied from 85 to 95% to generate rats with different degrees of hyperglycemia. Rats were classified according to their averaged blood glucose levels at 3 and 4 weeks post-Px. Values are means ± SE for eight sham (○), nine LPx (•), seven MPx (□), and six HPx (▪) rats. B: Representative comparison of HO-1, glutathione peroxidase, and A20 mRNA levels by semiquantitative multiplex RT-PCR in islets of sham, low hyperglycemic (LPx), mildly hyperglycemic (MPx), and highly hyperglycemic (HPx) rats. Cyclophilin and TBP were used as internal control genes. The averaged blood glucose values at 3 and 4 weeks post-Px are shown at the bottom. C: After normalization of the specific gene to its internal control gene, mRNA levels are expressed as a percent of sham. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham for each gene. BG, blood glucose; S, sham.
- FIG. 4.
Time course and reversibility of changes in islet gene expression after 90% Px. At 2 weeks post-Px, rats were either killed or randomly assigned to an untreated or a phlorizin-treated (0.8 g · kg−1 · day−1) group for the final 2 weeks. As control, sham animals received a similar volume of vehicle (1,2-propanediol). A: Effect of phlorizin treatment on blood glucose levels after Px. Values are means ± SE for three sham (○), four sham vehicle-treated (•), three hyperglycemic untreated Px (▪), and six phlorizin-treated Px (□) rats. B: Representative comparison of HO-1, glutathione peroxidase, and A20 mRNA levels by semiquantitative multiplex RT-PCR in islets of sham, sham vehicle-treated, hyperglycemic untreated Px (Px), and phlorizin-treated Px rats. Cyclophilin and TBP were used as internal control genes. Averaged blood glucose values (mg/dl) at 3 and 4 weeks post-Px are shown at bottom. C: Changes in mRNA levels at two (left panels) and 4 weeks (right panels) after Px. After normalization of the specific gene to its internal control gene, mRNA levels are expressed as a percent of sham. Data are means ± SE for the indicated number of experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham for each gene. BG, blood glucose; PxP, phlorizin-treated Px rats; S, sham; SP, sham vehicle-treated.
- FIG. 5.
Comparison by RT-PCR of stress gene mRNA levels in the β-cell-enriched central core of islets. The islet core tissue was extracted from pancreas sections of sham and hyperglycemic Px rats using laser capture microdissection. S, sham.
- FIG. 6.
Staining for HO-1 (A and B) and for insulin (red) and glucagon (green) (C and D) on pancreatic sections from a representative 4-week sham (A and C, same islet) and hyperglycemic Px rat (B and D, same islet).
- FIG. 7.
Increased NF-κB DNA binding activity in islets of hyperglycemic Px rats. At 4 weeks after surgery (90% Px and sham), nuclear extracts were obtained from the islets, and gel-shift assays were performed. A double-stranded oligonucleotide containing NF-κB consensus sequence was used as a binding probe (AGT TGA GGG GAC TTT CCC AGG C). In some of the binding assays, nonradioactive wild- or mutated-type competitor oligonucleotide (AGT TGA GGC GAC TTT CCC AGG C) or anti-NF-κB antibody (p65 or p50) were added. Similar results were obtained in two independent experiments.
- FIG. 8.
Increased 8-OHdG level in serum of hyperglycemic Px rats. At 4 weeks after surgery (90% Px and sham), 8-OHdG levels in serum were measured using an ELISA Kit. Data are expressed as means ± SE (n = 4). ***P < 0.001 vs. sham and LPx rats.
- FIG. 9.
Reduced sensitivity of Px islets to STZ-induced toxicity. Islets isolated from sham, Px, and phlorizin-treated Px rats were cultured in the absence (A) or presence (B) of 1 mmol/l STZ for 20 min and then without STZ for 24 h. Viability was assessed with acridine orange and PI and quantitated as the proportion of dead islet tissue (percent PI-stained area) using IP Lab Spectrum image analysis. Data are expressed as means ± SE for the indicated number of experiments. **P < 0.01 vs. STZ-treated sham. S, sham; SP, sham vehicle-treated.
Tables
- TABLE 1
Sequences of oligonucleotide primers and PCR conditions
Gene Name Size (bp) 5′ Oligonucleotide 3′ Oligonucleotide GenBank accession no. MgCl2 (mmol/l) dNTP (μmol/l) Primer (nmol/l) Annealing (°C) Cycles Control gene HO-I 307 ATG CCC CGC TCT ACT TCC GTG GGG TTG TCG ATC CTC J02722 1.5 160 500 62 27 Cyclophilin Glutathione peroxidase 301 GGT TTC CCG TGC AAT CAG GAA CAC CGT CTG GAC CTA CC X07365 1.5 80 200 59 27 TBP Cu/Zn-SOD 333 AAC TGA AGG CGA GCA TGG ATC ACA CCA CAA GCC AAGC M21060 1.5 80 200 56 23 α-Tubulin Mn-SOD 410 CAC CAC AGC AAG CAC CAC TCC CAC ACA TCA ATC CCC Y00497 1.5 160 80 62 26 TBP Catalase 173 GCT CCC AAC TAC TAC CCC AAC CGT TTC CTC TCC TCC TCA TTC M11670 1.5 160 100 62 27 α-Tubulin A20 497 TTT GAG CAA TAT GCG GAA AGC AGT TGT CCC ATT CGT CAT TCC U19463 2.0 80 400 55 30 TBP BCL-2 170 CCT GGC ATC TTC TCC TTC AGA AGT CAT CCC CAG CCC L14680 1.5 120 400 62 27 Cyclophilin HSP70 278 ACG CAG ACC TTC ACC ACC CGC TCG ATC TCC TCC TTG X75357 1.5 160 200 62 28 TBP NF-κB (subunit p65) 144 TTT CCC CTC ATC TTT CCC TC TGT GCT TCT CTC CCC AGG AF079314 1.5 160 400 62 26 TBP p53 295 GCT CAC TCC AGC TAC CCG ACC CAG CAA CTA CCA ACC C X13058 1.5 160 150 62 27 α-Tubulin Fas 322 TGA GCC TTG GAG ACG AAC ATC TTG GGG GCT GTT GTG D26112 1.5 160 400 62 30 TBP Cyclophilin 400 AAC CCC ACC GTG TTC TTC TGC CTT CTT TCA CCT TCC C M19533 1.5 120–160 40 62 TBP 190 ACC CCT CAC CAA TGA CTC CTA TG ATG ATG ACT GCA GCA AAT CGC D01034 1.5–2.0 80–160 100–200 55–62 α-Tubulin 451 CTC GCA TCC ACT TCC CTC ATG CCC TCA CCC ACG TAC J00797 1.5 160 200 56–62 - TABLE 2
Effect of in vivo dexamethasone treatment on body weight, blood glucose, and plasma insulin at 4 weeks after sham or Px
Sham saline Sham-Dex Px-saline Px-Dex n 3 3 3 3 Body weight (g) 316 ± 18 293 ± 20 308 ± 7 282 ± 13 Blood glucose (mg/dl) 103 ± 4 109 ± 2 168 ± 22* 327 ± 33**† Plasma insulin (ng/ml) 1.3 ± 0.1 2.3 ± 0.3* 1.6 ± 0.5 1.3 ± 0.5 -
Data are means ± SE. Body weight, blood glucose, and plasma insulin were measured 48 h after initial administration of dexamethasone (0.4 mg/kg, once daily for 2 consecutive days) or saline.
- *
* P < 0.05,
- †
† P < 0.01 vs. saline-treated sham rats;
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‡ P < 0.05 vs. saline-treated Px rats. Dex, dexamethasone.
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