Research ArticleDiabetes

Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes

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Science Translational Medicine  14 Jun 2017:
Vol. 9, Issue 394, eaah4477
DOI: 10.1126/scitranslmed.aah4477
  • Fig. 1. Effects of SFN on glucose production in H4IIE hepatoma cells.

    Glucose production (GP) was assessed during a 5-hour incubation in glucose-free buffer with pyruvate, l-lactate, and l-glutamine (GP-buffer). (A) GP in the presence or absence of 10 nM insulin (INS) in the GP-buffer assessed after 24-hour preincubation with or without SFN as indicated (n = 5). * denotes control (ctrl) cells versus SFN-treated cells; # denotes insulin-treated cells in the absence versus presence of SFN. (B) As in (A), with or without metformin (met) preincubation for 24 hours instead of SFN (n = 5). (C) GP assessed after 16 hours of pretreatment with 250 μM palmitate (palm) followed by 24 hours with or without 3 μM SFN (n = 4). (D) Representative immunoblot and summary statistics showing nuclear translocation of NRF2 protein after 1 hour of incubation with SFN at the doses indicated. Nucleoporin 62 (NUP62) was used as loading control (n = 3). (E) GP after knockdown of Nrf2 (Nrf2-KD) or treatment with a negative control siRNA followed by 24-hour preincubation with or without 3 μM SFN (n = 5). (F) mRNA expression of genes involved in gluconeogenesis after treatment with 3 μM SFN for 24 hours. Statistical analysis was performed using log2-transformed data (n = 4 to 6). (G) Representative immunoblot and summary statistics of PCK1 protein expression after knockdown of Nrf2 (Nrf2-KD) or treatment with a negative control siRNA followed by 24-hour preincubation with or without 3 μM SFN (n = 4). a.u., arbitrary units. (H) GP after knockdown of Pck1 (Pck1-KD) or treatment with a negative control siRNA. Cells were then treated with or without 3 μM SFN or 250 μM metformin for 24 hours as indicated (n = 5). * denotes control versus SFN, metformin, or insulin in cells treated with a negative control siRNA; # denotes control versus SFN, metformin, or insulin in Pck1-KD cells. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01.

  • Fig. 2. Effects of SFN in mouse hepatocytes and in rat models of diet-induced glucose intolerance.

    (A) Glucose production from primary mouse hepatocytes during 45 min. Cells were preincubated in the presence or absence of 500 μM bovine serum albumin–bound palmitate followed by 24-hour incubation with or without 3 μM SFN as indicated (n = 3). (B) Left: Fasting blood glucose in male Wistar rats before and after 15 weeks of HFD feeding with or without concomitant SFN treatment [SFN (2.5 mg/kg) three times per week; n = 9 per group]. Right: Longitudinal measurements of fasting glucose during the 15-week period. P value for the blood glucose for the SFN-treated compared to control-treated rats during the 3- to 15-week period is shown. (C) IPITT after the 15-week period for the same rats as in (B). (D to G) Insulin resistance (IR) and glucose tolerance assessed in the same animals as in (B) after an additional 5 weeks on 60% HFrD or HFD with or without concomitant SFN treatment. IPGTT (D) and IPITT (E) data from rats on 60% HFrD (n = 4 to 5 per group), and IPGTT (F) and IPITT (G) data from rats on 60% HFD (n = 4 to 5 per group). (H) OGTT data from male Wistar rats fed a low-fat control diet (ctrl diet) or a 60% HFD for 11 months before and after 14 days of treatment with SFN (5 mg/kg per day) or vehicle (n = 5 HFD ctrl, n = 6 HFD SFN, and n = 8 ctrl diet). (I) OGTT in male Wistar rats with diet-induced glucose intolerance (fed 60% HFrD for 6 months) after 10 days of treatment with or without SFN (10 mg/kg per day, ip) or metformin (300 mg/kg, po) as indicated. * denotes SFN (n = 6) versus vehicle (n = 7); # denotes metformin (n = 6) versus the corresponding vehicle (n = 8). (J) IPPTT on the same rats as in (I) after 9 to 12 days of treatment with or without SFN or metformin as indicated. One-sided t test was used for statistical analysis. * denotes SFN (n = 6) versus vehicle (n = 7); # denotes metformin (n = 6) versus the corresponding vehicle (n = 7). Data are means ± SEM. *P < 0.05; **P < 0.01; #P < 0.05; ##P < 0.01.

  • Fig. 3. Effects of SFN in mice with diet-induced diabetes.

    (A) IPGTT in male C57BL/6J mice fed a low-fat control diet or 60% HFD for 14 weeks treated with vehicle or SFN (0.5 or 10 mg/kg per day) for 4 weeks (n = 7 to 8 in each of the HFD groups, n = 5 to 6 in each of the control diet groups). (B) IPITT data from the mice in (A). (C) Triglyceride content in extracted liver tissue from the mice treated as in (A). (D) Gluconeogenetic rate in male C57BL/6J mice fed a 60% HFD for 12 weeks and treated with vehicle or SFN (10 mg/kg per day) for 4 weeks (n = 8 per group). The bars show data from the entire groups (average weight 40 ± 2 g for vehicle-treated and 39 ± 1 g for SFN-treated) as well as data for the three mice in each group with the highest body weight (heavy; weight of 42, 42, and 47 g for vehicle-treated and 39, 40, and 48 g for SFN-treated compared to an average weight of 38 ± 1 g for vehicle-treated and 37 ± 0.2 g for SFN-treated nonheavy mice). (E) Rate of disappearance of glucose (Rd) during clamp, reflecting whole-body insulin-stimulated glucose uptake, for the same mice as in (D). Data are means ± SEM. *P < 0.05; **P < 0.01.

  • Fig. 4. Effects of highly concentrated SFN provided as BSE in T2D patients.

    (A) Association between HbA1c at the start of the study (baseline) and treatment-induced change in HbA1c (ΔHbA1c) after 12 weeks in all patients (n = 50 placebo and n = 47 BSE). (B) Box plots showing median, upper and lower quartiles, and maximum and minimum values of treatment-induced change in fasting blood glucose and HbA1c in obese patients with dysregulated T2D (n = 9 placebo and n = 8 BSE). Circle denotes outlier. (C) Association between serum concentration of SFN (after 12 weeks of treatment) and treatment-induced change in fasting blood glucose in obese patients with dysregulated T2D (n = 8 BSE). (D) Association between treatment-induced change in fasting blood glucose and plasma triglyceride concentrations at the start of the study in all patients (n = 50 placebo and n = 47 BSE). (E) Association between treatment-induced change in fasting blood glucose and HOMA-IR at the start of the study in patients with dysregulated T2D (n = 18 placebo and n = 19 BSE). (F) Association between treatment-induced change in HbA1c and fatty liver index at the start of the study in patients with dysregulated T2D (n = 18 placebo and n = 19 BSE). *P < 0.05.

  • Table 1. Effects of 12 weeks of treatment with BSE on clinical variables in T2D patients.

    Data are means ± SD for patients with well-regulated (HbA1c ≤ 50 mmol/mol) and with dysregulated T2D (HbA1c > 50 mmol/mol) who are nonobese (BMI ≤ 30 kg/m2) or obese (BMI > 30 kg/m2). Data measured before treatment start (baseline) and after 12 weeks on placebo or BSE.

    TreatmentPhenotypeTimeWell-regulated T2DDysregulated T2D
    Nonobese (n = 28)Obese (n = 31)Nonobese (n = 21)Obese (n = 17)
    PlaceboHbA1c (mmol/mol)Baseline46.4 ± 2.845.6 ± 3.454.6 ± 3.256.3 ± 7.5
    12 weeks47.5 ± 4.146.3 ± 3.454.9 ± 4.456.6 ± 9.2
    Fasting P-glucose (mM)Baseline7.51 ± 0.917.26 ± 0.868.84 ± 0.848.33 ± 1.03
    12 weeks7.55 ± 0.847.29 ± 0.879.08 ± 1.318.91 ± 1.75
    P-glucose 120 min (mM)Baseline13.77 ± 3.1611.55 ± 2.5817.00 ± 3.1415.81 ± 2.50
    12 weeks13.27 ± 3.5811.22 ± 2.0217.10 ± 3.8215.28 ± 3.42
    BMI (kg/m2)Baseline27.6 ± 1.933.3 ± 2.428.0 ± 1.333.1 ± 2.0
    12 weeks27.8 ± 1.933.4 ± 2.528.1 ± 1.533.0 ± 2.1
    HOMA-IR (mM × mU/liter)Baseline1.75 ± 0.782.73 ± 1.502.36 ± 1.524.32 ± 1.80
    12 weeks1.83 ± 0.863.04 ± 2.102.52 ± 1.705.11 ± 3.30
    ISIBaseline3.39 ± 1.592.47 ± 1.903.48 ± 2.691.60 ± 0.73
    12 weeks3.70 ± 2.042.55 ± 2.083.41 ± 2.391.67 ± 0.79
    Fatty liver indexBaseline60.2 ± 18.183.1 ± 13.958.0 ± 18.787.9 ± 10.8
    12 weeks58.0 ± 17.781.2 ± 16.657.5 ± 22.287.9 ± 10.9
    P-triglycerides (mM)Baseline1.49 ± 0.651.39 ± 0.561.50 ± 0.491.88 ± 0.89
    12 weeks1.40 ± 0.671.26 ± 0.421.40 ± 0.551.94 ± 0.89
    BSEHbA1c (mmol/mol)Baseline45.7 ± 3.246.1 ± 3.055.7 ± 6.057.1 ± 6.6
    12 weeks46.9 ± 3.546.7 ± 2.757.3 ± 5.253.4 ± 6.8
    Fasting P-glucose (mM)Baseline7.49 ± 1.167.34 ± 0.948.61 ± 1.418.58 ± 1.60
    12 weeks7.91 ± 1.687.60 ± 1.368.39 ± 1.248.15 ± 1.26
    P-glucose 120 min (mM)Baseline13.99 ± 4.2213.33 ± 3.8415.52 ± 3.2615.64 ± 3.63
    12 weeks13.83 ± 4.2713.36 ± 3.1716.51 ± 3.0915.41 ± 3.61
    BMI (kg/m2)Baseline28.1 ± 1.432.9 ± 1.827.8 ± 1.333.2 ± 2.1
    12 weeks28.2 ± 1.433.4 ± 2.327.7 ± 1.333.1 ± 2.3
    HOMA-IR (mM × mU/liter)Baseline1.76 ± 0.762.46 ± 1.422.32 ± 1.773.02 ± 1.34
    12 weeks2.44 ± 0.883.08 ± 1.502.20 ± 1.623.05 ± 0.87
    ISIBaseline2.78 ± 0.892.00 ± 0.844.08 ± 2.092.32 ± 1.03
    12 weeks2.40 ± 1.001.92 ± 0.763.92 ± 1.982.28 ± 1.05
    Fatty liver indexBaseline60.6 ± 18.783.2 ± 10.461.5 ± 18.887.7 ± 9.8
    12 weeks62.9 ± 18.485.6 ± 10.362.4 ± 18.287.2 ± 7.6
    P-triglycerides (mM)Baseline1.42 ± 0.641.35 ± 0.401.87 ± 1.331.50 ± 0.53
    12 weeks1.46 ± 0.651.42 ± 0.422.10 ± 1.231.40 ± 0.48

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/394/eaah4477/DC1

    Materials and Methods

    Fig. S1. Effect of SFN on apoptosis, insulin signaling, and OCR in H4IIE cells.

    Fig. S2. Effect of SFN on the 50-gene hepatic disease signature.

    Fig. S3. Effect of BSE on H4IIE cells and on mice fed an HFD.

    Table S1. The 50-gene liver disease signature for T2D.

    Table S2. List of compounds used in the analysis (see separate Excel file).

    Table S3. Rank order of compounds (see separate Excel file).

    Table S4. Expression of enzymes involved in gluconeogenesis.

    Table S5. Adverse effects in patients treated with BSE or placebo for 12 weeks.

    Table S6. Characteristics of patients discontinuing the study.

    References (6169)

  • Supplementary Material for:

    Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes

    Annika S. Axelsson, Emily Tubbs, Brig Mecham, Shaji Chacko, Hannah A. Nenonen, Yunzhao Tang, Jed W. Fahey, Jonathan M. J. Derry, Claes B. Wollheim, Nils Wierup, Morey W. Haymond, Stephen H. Friend, Hindrik Mulder, Anders H. Rosengren*

    *Corresponding author. Email: anders.rosengren{at}gu.se

    Published 14 June 2017, Sci. Transl. Med. 9, eaah4477 (2017)
    DOI: 10.1126/scitranslmed.aah4477

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Effect of SFN on apoptosis, insulin signaling, and OCR in H4IIE cells.
    • Fig. S2. Effect of SFN on the 50-gene hepatic disease signature.
    • Fig. S3. Effect of BSE on H4IIE cells and on mice fed an HFD.
    • Table S1. The 50-gene liver disease signature for T2D.
    • Legends for tables S2 and S3
    • Table S4. Expression of enzymes involved in gluconeogenesis.
    • Table S5. Adverse effects in patients treated with BSE or placebo for 12 weeks.
    • Table S6. Characteristics of patients discontinuing the study.
    • References (6169)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2. List of compounds used in the analysis (see separate Excel file).
    • Table S3. Rank order of compounds (see separate Excel file).

    Download Tables S2 and S3