Adenoviral Gene Transfer of PLD1-D4 Enhances Insulin Sensitivity in Mice by Disrupting Phospholipase D1 Interaction with PED/PEA-15

<div><p>Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity <i>in vivo</i> has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg _ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.</p> </div

Effect of D4 on PKCalpha and zeta activation in Tg_Ped/pea-15mice.

<p>PKCalpha (<b>A</b>) and zeta (<b>B</b>) activations were determined in the skeletal muscle tissues from Tg_Ped/pea-15mice at one week post Ad-D4 or Ad-GFP infection. Tg_Ped/pea-15 or wild type (Wt) mice injected with PBS (vehicle) were used as control. For the experiment, mice were fasted over night and then i.p. injected or not with insulin (10 U/kg body weight) 10 min before determination. <b>A)</b> The bar graph represents the densitometric quantization of phospho-PKCalpha in three independent immunoblots. ***<i>p</i><0.001 vs. Wt. <b>B)</b> The corresponding blots show the levels of PKCzeta (total and phosphorilated forms) and tubulin in mice as indicated. Blots are representative of three independent experiments.</p

Metabolic characteristics and food intake of HFD and STD fed C57Bl/6 mice.

<p>Mice were analysed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060555#s2" target="_blank">Materials and Methods</a>. Data are the means ± SEM of determinations in 12 STD and 12 HFD fed C57Bl/6 mice on the following conditions: baseline, upon 11 week of diet (w11), upon w11 treated with Ad-GFP and upon w11 treated with Ad-D4. HFD w11 vs. STD w11 mice;</p>a<p><i>p</i><0.01 and</p>b<p><i>p</i><0.001; HFD w11 Ad-GFP vs. STD w11 mice;</p>c<p><i>p</i><0.001; HFD w11 Ad-D4 vs. STD w11 mice;</p>d<p><i>p</i><0.001; HFD w11 Ad-D4 vs. HFD w11 Ad-GFP mice;</p>e<p><i>p</i><0.001; n.a., not available.</p

Metabolic characteristics of TgPed/pea-15 mice.

<p>Data are means ± SEM of determinations in at least 10 mice per group. Vehicle TgPed/pea-15 mice vs. Vehicle Wild Type mice;</p>a<p><i>P</i><0.001. Ad-GFP TgPed/pea-15 mice vs. Vehicle Wild Type mice;</p>b<p><i>P</i><0.001. Ad-GFP TgPed/pea-15 mice vs. Vehicle TgPed/pea-15 mice;</p>c<p><i>P</i><0.001. Ad-D4 TgPed/pea-15 mice vs. Vehicle Wild Type mice;</p>d<p><i>P</i><0.001. Ad-D4 TgPed/pea-15 mice vs. Vehicle TgPed/pea-15 mice;</p>e<p><i>P</i><0.01 and</p>f<p><i>P</i><0.001. Ad-D4 TgPed/pea-15 mice vs. Ad-GFP TgPed/pea-15 mice;</p>g<p><i>P</i><0.01 and</p>h<p><i>P</i><0.001.</p

Activation of caspase-3 and PARP cleavage.

<p>A) Caspase-3 activation. Cells were grown on a glass coverslips and treated or not with 70 µM hydrogen peroxide for 48 hours. After fixation and permeabilization, cells were processed for anticaspase-3 antibody (in red). Arrows indicate cells stained with antibody to the active form of caspase-3. The total number of cells is visualized by the background staining. The results shown are representative of 3 independent experiments. B) PARP cleavage. Cells were treated or not for 48 hours with 70 µM hydrogen peroxide, as indicated. Whole-cell extracts (30 µg) were analyzed by Western blot with anti-PARP antibody. Full-length PARP (113 kDa) and the cleaved fragment (89 kDa) are indicated by arrowheads. Tubulin was used as loading control (n = 3). A representative image is shown.</p

Insulin sensitivity in STD and HFD fed C57BL/6 mice.

<p><b>A)</b> Immunoblots of total protein lysates from skeletal muscle homogenates of C57BL/6 mice fed with standard (STD) or high fat (HFD) diets after infection with Ad-GFP or with Ad-D4. The upper blot was probed with anti-GFP antibody, while the bottom blot was probed with anti-Tubulin antibody. <b>B)</b> Insulin Tolerance Test (ITT) and (<b>C</b>) Area Under the Curve (AUC) glucose in C57BL/6 mice fed with standard (STD, square) or high fat (HFD, circle) diets after infection with Ad-GFP (white and dark gray) or with Ad-D4 (black and light gray). For each experiment, values are expressed as means ± SEM of determinations in at least eight mice per group. ***p<0.001 vs Ad-GFP treated STD fed C57BL/6 mice. ##p<0.01, vs Ad-GFP treated HFD fed C57BL/6 mice.</p

D4 mRNA expression in Tg_Ped/pea-15mice.

<p><i>D4</i> mRNA expression was determined by quantitative Real Time RT-PCR analysis of total RNA isolated from liver (<b>A</b>), pancreas (<b>B</b>), and tibialis skeletal muscle tissue (<b>C</b>) of Tg_Ped/pea-15mice at one week post Ad-D4 or Ad-GFP infection. GAPDH was used as housekeeping gene. mRNA levels in Ad-D4 treated Tg_Ped/pea-15mice are relative expression units to those in Ad-GFP treated Tg_Ped/pea-15mice used as control (mean ± SEM; n = 3). ***<i>p</i><0.001 vs Ad-GFP treated Tg_Ped/pea-15.</p

Apoptosis in isolated mice islets.

<p>Mouse pancreatic islets were treated or not with 10 µM hydrogen peroxide for 16 hours. After this, histone-associated DNA fragments were quantified by ELISA to evaluate apoptotic cell death. Each column represents the mean ± SE from three separate experiments. ***<i>p</i><0.001.</p

A) <i>HA-D4 transfection</i>.

<p>pcDNA3-HA-D4 and the control plasmid pcDNA3-HA were transfected in both Ins-1E_PED/PEA-15 and in Ins-1E_CTRL cells, and D4 expression was analyzed by qualitative PCR. <b>B) </b><b><i>Apoptosis.</i></b> Cells were incubated with 70 µM hydrogen peroxide as indicated. After 48 hours, cells were harvested and apoptosis was quantitated by evaluating the level of DNA fragmentation using the Roche Cell Death Detection ELISAPLUS. Data are the mean value of four identical wells. Values represent the mean ±SD of three independent experiments. ***<i>p</i><0.001.</p

Glucose uptake in skeletal muscle tissues of Tg_Ped/pea-15mice.

<p>Ex vivo Glucose uptake into gastrocnemius (<b>A</b>), tibialis (<b>B</b>) and quadriceps (<b>C</b>) skeletal muscle of Tg_Ped/pea-15mice treated with vehicle (black column) or after infection with Ad-GFP (light grey column) or with Ad-D4 (dark grey column), and wild type mice (Wt, white column) used as controls. Values are expressed as means ± SEM of determinations in at least five mice per group. ***<i>p</i><0.001 vs Wt.</p