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Islet Studies

Increased Expression of Antioxidant and Antiapoptotic Genes in Islets That May Contribute to β-Cell Survival During Chronic Hyperglycemia

  1. D. Ross Laybutt1,
  2. Hideaki Kaneto1,
  3. Wendy Hasenkamp1,
  4. Shane Grey2,
  5. Jean-Christophe Jonas1,
  6. Dennis C. Sgroi3,
  7. Adam Groff1,
  8. Christiane Ferran2,
  9. Susan Bonner-Weir1,
  10. Arun Sharma1 and
  11. Gordon C. Weir1
  1. 1Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Boston, Massachusetts
  2. 2Immunobiology Research Center, Beth Israel Deaconess Medical Center, Boston, Massachusetts
  3. 3Molecular Pathology Unit, Massachusetts General Hospital, Boston, Massachusetts
    Diabetes 2002 Feb; 51(2): 413-423. https://doi.org/10.2337/diabetes.51.2.413
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    • FIG. 1.
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      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.
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      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.
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      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.
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      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.
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      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.
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      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.
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      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.
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      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.
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      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

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    • TABLE 1

      Sequences of oligonucleotide primers and PCR conditions

      Gene NameSize (bp)5′ Oligonucleotide3′ OligonucleotideGenBank accession no.MgCl2 (mmol/l)dNTP (μmol/l)Primer (nmol/l)Annealing (°C)CyclesControl gene
      HO-I307ATG CCC CGC TCT ACT TCCGTG GGG TTG TCG ATC CTCJ027221.51605006227Cyclophilin
      Glutathione peroxidase301GGT TTC CCG TGC AAT CAGGAA CAC CGT CTG GAC CTA CCX073651.5802005927TBP
      Cu/Zn-SOD333AAC TGA AGG CGA GCA TGGATC ACA CCA CAA GCC AAGCM210601.5802005623α-Tubulin
      Mn-SOD410CAC CAC AGC AAG CAC CACTCC CAC ACA TCA ATC CCCY004971.5160806226TBP
      Catalase173GCT CCC AAC TAC TAC CCC AACCGT TTC CTC TCC TCC TCA TTCM116701.51601006227α-Tubulin
      A20497TTT GAG CAA TAT GCG GAA AGCAGT TGT CCC ATT CGT CAT TCCU194632.0804005530TBP
      BCL-2170CCT GGC ATC TTC TCC TTCAGA AGT CAT CCC CAG CCCL146801.51204006227Cyclophilin
      HSP70278ACG CAG ACC TTC ACC ACCCGC TCG ATC TCC TCC TTGX753571.51602006228TBP
      NF-κB (subunit p65)144TTT CCC CTC ATC TTT CCC TCTGT GCT TCT CTC CCC AGGAF0793141.51604006226TBP
      p53295GCT CAC TCC AGC TAC CCGACC CAG CAA CTA CCA ACC CX130581.51601506227α-Tubulin
      Fas322TGA GCC TTG GAG ACG AACATC TTG GGG GCT GTT GTGD261121.51604006230TBP
      Cyclophilin400AAC CCC ACC GTG TTC TTCTGC CTT CTT TCA CCT TCC CM195331.5120–1604062
      TBP190ACC CCT CAC CAA TGA CTC CTA TGATG ATG ACT GCA GCA AAT CGCD010341.5–2.080–160100–20055–62
      α-Tubulin451CTC GCA TCC ACT TCC CTCATG CCC TCA CCC ACG TACJ007971.516020056–62
    • TABLE 2

      Effect of in vivo dexamethasone treatment on body weight, blood glucose, and plasma insulin at 4 weeks after sham or Px

      Sham salineSham-DexPx-salinePx-Dex
      n3333
      Body weight (g)316 ± 18293 ± 20308 ± 7282 ± 13
      Blood glucose (mg/dl)103 ± 4109 ± 2168 ± 22*327 ± 33**†
      Plasma insulin (ng/ml)1.3 ± 0.12.3 ± 0.3*1.6 ± 0.51.3 ± 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;

      • ‡ P < 0.05 vs. saline-treated Px rats. Dex, dexamethasone.

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    Increased Expression of Antioxidant and Antiapoptotic Genes in Islets That May Contribute to β-Cell Survival During Chronic Hyperglycemia
    D. Ross Laybutt, Hideaki Kaneto, Wendy Hasenkamp, Shane Grey, Jean-Christophe Jonas, Dennis C. Sgroi, Adam Groff, Christiane Ferran, Susan Bonner-Weir, Arun Sharma, Gordon C. Weir
    Diabetes Feb 2002, 51 (2) 413-423; DOI: 10.2337/diabetes.51.2.413

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    Increased Expression of Antioxidant and Antiapoptotic Genes in Islets That May Contribute to β-Cell Survival During Chronic Hyperglycemia
    D. Ross Laybutt, Hideaki Kaneto, Wendy Hasenkamp, Shane Grey, Jean-Christophe Jonas, Dennis C. Sgroi, Adam Groff, Christiane Ferran, Susan Bonner-Weir, Arun Sharma, Gordon C. Weir
    Diabetes Feb 2002, 51 (2) 413-423; DOI: 10.2337/diabetes.51.2.413
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