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Excessive caloric intake acutely causes oxidative stress, GLUT4 carbonylation, and insulin resistance in healthy men

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Science Translational Medicine  09 Sep 2015:
Vol. 7, Issue 304, pp. 304re7
DOI: 10.1126/scitranslmed.aac4765
  • Fig. 1. Development of insulin resistance in response to overnutrition in healthy men.

    (A) Daily basal concentrations of plasma glucose, insulin, and FFA, as well as HOMA-IR during 7 days of overnutrition (n =6). (B) Glucose, insulin, and FFA concentrations, as well as GIRs, during euglycemic-hyperinsulinemic clamps performed immediately before and after overnutrition. Inserted box in (B) shows GIR before and after overnutrition in six individual subjects. Shown are means ± SE. Table S1 shows individual data for all study subjects. *P < 0.05, **P < 0.01 compared to day 1. Day 1, before overnutrition.

  • Fig. 2. Connection between overnutrition-induced insulin resistance and oxidative stress.

    (A) Individual daily basal plasma cytokine concentrations of the six study participants during the 7 days of overnutrition. (B) Unfolded protein response (UPR) marker mRNA and proteins in subcutaneous adipose tissue biopsies obtained from the six study subjects immediately before and after overnutrition. Shown are means ± SE. (C) Daily urinary 8-iso-PGF2α concentration and HOMA-IR during 7 days of overnutrition. n = 6; shown are means ± SE. Table S2 shows individual data for all study subjects shown in (B) and (C). *P < 0.05 compared to day 1. ATF4, activating transcription factor 4; BIP, immunoglobulin binding protein; CHOP, C/EBP homology protein; CNX, calnexin; CRT, calreticulin; XBP-1s, X-box binding protein 1s; GRP94, 94 kDa glucose-regulated protein.

  • Fig. 3. Overnutrition-induced ROS production and posttranslational modifications of adipose tissue proteins.

    Two-dimensional (2D) gel electrophoresis oxyblot analysis of carbonylated proteins before (left panel) and after (right panel) overnutrition. The extent of posttranslational modifications, as indicated by carbonylation, is shown in table S4. Results were obtained from pooled lysates of the six individual fat biopsies. pI, isoelectric point.

  • Fig. 4. Identification of GLUT4 posttranslational modifications in response to overnutrition.

    (A) 2D gel electrophoresis Western blots showing multiple isoforms of GLUT4 before (left panel) and after (right panel) excess nutrient uptake. The posttranslational modifications associated with these isoforms were identified by mass spectroscopy analysis (table S6). Results were obtained from pooled lysates of six individual fat biopsies. IgG, immunoglobulin G. (B) Homology-based modeling of GLUT4 structure showing the localization of posttranslationally modified amino acids (R246, R265, and K264) in the cytosolic helices. The amino acids in red were carbonylated, and the amino acids in blue contained 4-HNE adduction. (C) Space-filling model of GLUT4 showing the close proximity of the modified amino acids to the glucose transport channel (marked with red arrowheads). (D) GLUT4 putative topology showing the 12 transmembrane regions (TM) and the intracellular helix (IC). The posttranslational modifications are indicated by red stars (carbonylation) and blue triangle (HNE).

  • Table 1. Study subjects.

    BMI, body mass index; FM, fat mass; FFM, fat-free mass; NS, not significant.

    Day 1Day 7ChangeP
    Age (years)50.0 ± 1.450.2 ± 3.5
    Gender (male/female)6/06/0
    Height (cm)174 ± 2174 ± 2
    Weight (kg)79.0 ± 2.782.5 ± 2.8+3.5 ± 0.3<0.0001
    BMI (kg/m2)25.9 ± 0.927.1 ± 1.0+1.13 ± 0.11<0.0001
    FM (kg)15.1 ± 1.618.6 ± 1.9+3.5 ± 1.0<0.0001
    FFM (kg)63.9 ± 1.363.9 ± 1.6+0.0 ± 0.8NS
    REE (kcal/24 hours)1482 ± 991719 ± 127237 ± 800.045
  • Table 2. Energy balance.

    TCI, total caloric intake.

    Daily caloric intake (kcal/24 hours)6,206 ± 256
    TCI (kcal/7 days)42,955 ± 805
    REE (kcal/7days)11,248 ± 510
    TRF (kcal/7 days)2,977 ± 61
    Total energy expenditure (TEE = REE + TRF) (kcal/7days)14,226 ± 487
    Total caloric excess (TCI − TEE)28,729 ± 1,140
    Expected fat gain (kg)3.19 ± 0.13
    Observed fat gain (kg)3.50 ± 0.3

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/304/304re7/DC1

    Table S1. Individual daily measurements for Fig. 1A and individual clamp measurements for Fig. 1B.

    Table S2. Individual measurements for Fig. 2 (B and C).

    Table S3. Differentially expressed proteins in adipose tissue in response to overnutrition identified by GeLC-MS/MS (provided as an Excel file).

    Table S4. Overnutrition-induced increases in proteins related to generation or detoxification of ROS.

    Table S5. Identification of ROS-related posttranslational modifications in adipose tissue in response to overnutrition (provided as an Excel file).

    Table S6. Overnutrition-induced modifications in GLUT4 immunoprecipitated from adipose tissue protein extracts.

    Table S7. Primer sets used for RT-PCR studies.

  • Supplementary Material for:

    Excessive caloric intake acutely causes oxidative stress, GLUT4 carbonylation, and insulin resistance in healthy men

    Guenther Boden,* Carol Homko, Carlos A. Barrero, T. Peter Stein, Xinhua Chen, Peter Cheung, Chiara Fecchio, Sarah Koller, Salim Merali*

    *Corresponding author. E-mail: bodengh{at}tuhs.temple.edu (G.B.); smerali{at}temple.edu (S.M.)

    Published 9 September 2015, Sci. Transl. Med. 7, 304re7 (2015)
    DOI: 10.1126/scitranslmed.aac4765

    This PDF file includes:

    • Table S1. Individual daily measurements for Fig. 1A and individual clamp measurements for Fig. 1B.
    • Table S2. Individual measurements for Fig. 2 (B and C).
    • Legend for table S3
    • Table S4. Overnutrition-induced increases in proteins related to generation or detoxification of ROS.
    • Legend for table S5
    • Table S6. Overnutrition-induced modifications in GLUT4 immunoprecipitated from adipose tissue protein extracts.
    • Table S7. Primer sets used for RT-PCR studies.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Differentially expressed proteins in adipose tissue in response to overnutrition identified by GeLC-MS/MS (provided as an Excel file).
    • Table S5. Identification of ROS-related posttranslational modifications in adipose tissue in response to overnutrition (provided as an Excel file).

    [Download Tables S3 and S5]

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