# Sunflower Oil Supplementation Has Proinflammatory Effects and Does Not Reverse Insulin Resistance in Obesity Induced by High-Fat Diet in C57BL/6 Mice

Authors: Laureane Nunes Masi; Amanda Roque Martins; José César Rosa Neto; Cátia Lira do Amaral; Amanda Rabello Crisma; Marco Aurélio Ramirez Vinolo; Edson Alves de Lima Júnior; Sandro Massao Hirabara; Rui Curi
Journal: Journal of Biomedicine and Biotechnology
 (2012)

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## Abstract

High consumption of polyunsaturated fatty acids, such as sunflower oil has been associated to beneficial effects in plasma lipid profile, but its role on inflammation and insulin resistance is not fully elucidated yet. We evaluated the effect of sunflower oil supplementation on inflammatory state and insulin resistance condition in HFD-induced obese mice. C57BL/6 male mice (8 weeks) were divided in four groups: (a) control diet (CD), (b) HFD, (c) CD supplemented with n-6 (CD + n-6), and (d) HFD supplemented with n-6 (HFD + n-6). CD + n-6 and HFD + n-6 were supplemented with sunflower oil by oral gavage at 2 g/Kg of body weight, three times per week. CD and HFD were supplemented with water instead at the same dose. HFD induced whole and muscle-specific insulin resistance associated with increased inflammatory markers in insulin-sensitive tissues and macrophage cells. Sunflower oil supplementation was not efficient in preventing or reducing these parameters. In addition, the supplementation increased pro-inflammatory cytokine production by macrophages and tissues. Lipid profile, on the other hand, was improved with the sunflower oil supplementation in animals fed HFD.  In conclusion, sunflower oil supplementation improves lipid profile, but it does not prevent or attenuate insulin resistance and inflammation induced by HFD in C57BL/6 mice.


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## Body

## 1. Introduction

Occidental    diet    is    characterized    by    high    caloric    intake,    mainly    saturated    fatty    acids    and    glucose    consumption,    contributing    to    the    development    of    obesity    and    insulin    resistance.    In    the    past    15    years,    obesity    has    been    associated    to    chronic    inflammation    in    several    tissues    and    cells,    such    as    liver,    adipose    tissue,    skeletal    muscle,    and    immune    cells.In    fact,    chronic    and    subclinical    “low-grade”    inflammatory    state    is    a    hallmark    of    obesity,    and    this    condition    has    been    proposed    to    play    a    central    role    in    the    development    of    insulin    resistance,    type    2diabetes    mellitus,    and    steatosis    [1].    During    the    past    decades,    the    prevalence    of    obesity    has    enormously    increased    worldwide    [2].    A    possible    interaction    factor    between    metabolic    disorders    and    inflammation    is    the    disruption    in    lipid    metabolism    caused    by    high    levels    of    saturated    fatty    acids    from    high-fat    and    caloric    diet    [3, 4].C57BL/6    mice    develop    obesity,    insulin    resistance    (IR),diabetes    mellitus,    advanced    fatty    liver,    and    fatty    pancreatic    diseases    when    submitted    to    a    high-fat    diet    (HFD),    mainly    enriched    with    saturated    fatty    acids    [5].    The    composition    of    fatty    acids    on    diet    can    be    an    important    modulator    of    lipid    metabolism    and    inflammation.    Some    studies    have    showed    that    an    increase    in    circulating    levels    of    mono-    or    polyunsaturated    fatty    acids    improves    insulin    sensitivity    and    ameliorates    hepatic    steatosis    [6, 7].    Sunflower    oil    is    rich    in    mono-    (MUFA)    and    polyunsaturated    fatty    acids    (PUFA),    mainly    linoleic    acid,    a    member    of    the    n-6    family.Diet    enriched    with    sunflower    oil    decreases    plasma    triacylglycerol    and    has    beneficial    effects    on    plasma    lipid    profile    [8].    Moreover,    acute    administration    of    sunflower    oil    in    rats    potentially    prevents    the    gastric    damages    generated    by    indomethacin    administration    through    increased    anti-inflammatory    response    [9].    However,    mice    fed    with    sunflower    oil-enriched    diet    present    increased    IL-6    (a    pro-inflammatory    cytokine)    levels    in    white    adipose    tissue[8].    Thus,    further    studies    are    required    to    adequately    determine    the    pro-    or    anti-inflammatory    effects    of    n-6    fatty    acids.Adipose    tissue,    skeletal    muscle,    liver,    and    immune    cells    have    been    identified    as    critical    targets    for    the    disruption    in    metabolic    and    inflammatory    signaling    in    obesity.    The    imbalance    between    metabolic    alterations    and    inflammatory    signaling    pathways    leads    to    insulin    resistance,    hepatic    steatosis,    and    related    diseases    [4, 10].In    this    study,    we    evaluated    the    effect    of    sunflower    oil    supplementation    on    inflammation    and    insulin    sensitivity    in    mice    fed    a    balanced    diet    and    a    HFD    (rich    in    saturated    fatty    acids).    The    protecting    or    potentiating    effect    of    sunflower    oil    supplementation    on    insulin    resistance    and    inflammation    was    investigated.    Whole    body    (Insulin    Tolerance    Test—ITT    and    Glucose    Tolerance    Test—GTT)    and    skeletal    muscle    (glucose    uptake    and    metabolism)    insulin    sensitivity    was    examined.    Inflammatory    markers    were    investigated    in    liver,    adipose    tissue,    skeletal    muscle,    and    peritoneal    macrophages.    The    strategy    used    was    to    investigate    the    effect    of    sunflower    oil    supplementation    in    mice    fed    a    balanced    diet    and    a    HFD.    Under    this    last    condition,    a    clear    inflammatory    state    is    established    and    so    a    possible    anti-inflammatory    effect    could    be    pronounced.


## 2. Materials    and    Methods

### 2.1. Animals

All    animal    studies    were    performed    according    to    protocols    approved    by    the    Animal    Care    and    Use    Committee    from    the    Institute    of    Biomedical    Sciences,    University    of    São    Paulo.    C57BL/6    male    mice    (8    weeks-old)    were    used    for    the    study.    Animals    were    housed    in    a    room    with    light-dark    cycle    of    12-12 h    and    temperature    of23±2°C.    Animals    were    divided    in    four    groups:    (a)    control    diet    (CD),    (b)    high-fat    diet    (HFD),    (c)    control    diet    supplemented    with    n-6    (CD    +    n-6),    and    (d)    high-fat    diet    supplemented    with    n-6    (HFD    +    n-6).    During    the    first    4    weeks    preceding    the    induction    of    obesity    by    HFD,    all    the    four    experimental    groups    were    fed    ad    libitum    with    a    control    diet    (76%    carbohydrates,    9%    fat,    15%    proteins).    CD    +    n-6    and    HFD    +    n-6    were    supplemented    with    sunflower    oil    (n-6    PUFA    source)    by    oral    gavage    at    2 g    per    Kg    of    body    weight,    three    times    per    week,    during    12    weeks.    This    dosage    of    oil    was    chosen    based    on    previous    studies    using    different    oils    from    our    group    and    others    [11–13].    CD    and    HFD    received    water    at    the    same    dose.


### 2.2. HFD-Induced    Obesity    and    Insulin    Resistance

After    the    first    4    weeks,    animals    from    the    HFD    and    HFD    +    n-6    groups    received    high-fat    diet    (26%    carbohydrates,    59%    fat,    15%    proteins)    during    the    next    8    weeks.    CD    and    CD    +    n-6    groups    remained    on    the    control    diet.    Supplementation    with    n-6    (sunflower    oil,    2 g/Kg    b.w.)    was    kept    until    the    end    of    the    12    weeks.


### 2.3. Glucose    and    Insulin    Tolerance    Tests

Tolerances    to    glucose    (GTT)    and    to    insulin    (ITT)    were    evaluated    after    6 h    fasting.    For    GTT,    mice    were    intraperitoneally    injected    (i.p.)    with    glucose    (2 g/Kg    body    weight).    Blood    glucose    measurements    were    performed    at    0,    15,    30,    45,    60,    and    90 min    after    glucose    injection.    Glucose    concentration    versus    time    was    plotted    and    the    area    under    the    curve    (AUC)    was    calculated    for    each    animal.    For    ITT,    animals    were    i.p.    injected    with    insulin    (Humulin    R,    Lilly,    0.75 UI/kg    b.w.)    and    glucose    measurements    were    performed    at    0,    10,    20,    30,    40,    50,    and    60 min    after    injection.    Glucose    concentration    versus    time    was    plotted    and    the    glucose    lowering    rate    was    calculated.In    both    tests,    blood    samples    were    collected    from    the    tail    vein.    For    GTT,    serum    glucose    was    measured    by    colorimetric    assay    commercially    available    (PAP    Liquiform    Glucose,    Labtest)    and    for    ITT,    glucose    was    measured    by        using    glucometer    (One    Touch    Ultra,    Johnson    &    Johnson).


### 2.4. Serum    Parameters    Analysis

After    6    hours    fasting,    animals    were    anesthetized    and    blood    was    collected    by    puncturing    the    orbital    plexus.    Serum    glucose,    triacylglycerol,    total    cholesterol,    LDL-cholesterol,    and    HDL-cholesterol    were    determined    by    colorimetric    assays    (Labtest    Diagnostics,    Lagoa    Santa,    MG,    Brazil).


### 2.5. Responsiveness    to    Insulin    in    Isolated    Soleus    Muscle

Animals    were    euthanized    by    cervical    dislocation    and    soleus    muscles    rapidly    and    carefully    isolated,    weighed    (8–10 mg),    attached    to    stainless    steel    clips    to    maintain    resting    tension,    and    preincubated    for    30 min,    at    37°C,    in    Krebs-Ringer    bicarbonate    buffer    (KRBB)    containing    5.6 mM    glucose    and    1%    bovine    serum    albumin    (BSA),    pH    7.4,    pregassed    for    30 min    with    95%    O2/5%    CO2,    with    agitation    at    100    oscillations    per    min.    After    this    period,    muscles    were    transferred    to    other    vials    containing    the    same    buffer,    but    added    of    0.3 μCi/mL    D-[U-14C]-glucose    and    0.2 μCi/mL    2-deoxy-D-[2,6-3H]-glucose.    Phenylethylamine    (0.2 mL),    diluted    1 : 1 v/v    in    methanol,    was    added    into    a    separate    compartment    for    14CO2    adsorption.    Incubation    was    then    performed    for    1 h    under    similar    conditions,    in    the    absence    or    presence    of    7 nM    insulin.    Gasification    with    95%    O2/5%    CO2    was    maintained    during    the    pre-incubation    and    the    first    15 min    of    the    incubation    period    and    then    stopped.After    the    incubation    period,    muscles    were    briefly    washed    in    cold    KRBB    at    4°C,    dried    on    filter    paper    and    frozen    in    liquid    N2.    Samples    were    processed    for    measurements    of    uptake    of    2-deoxy-D-[2,6-3H]-glucose,    D-[14C]-glucose    incorporation,    [14C]-glycogen    synthesis,    and    decarboxylation    of    D-[14C]-glucose,    according    to    methods    described    by    Challiss    et    al.    [14],    Espinal    et    al.    [15]    and    Leighton    et    al.    [16],    respectively,    and    routinely    have    been    used    by    our    group    [11, 17–22].


### 2.6. Analysis    of    Inflammatory    Parameters

#### 2.6.1. Tissue    Cytokine    and    Adipokine    Content    Measurements

Mice    were    euthanized    on    CO2    chamber    and    visceral    adipose    tissue    (epididymal,    retroperitoneal,    and    mesenteric),    liver,    gastrocnemius    muscle,    and    peritoneal    macrophages    rapidly    collected.    About    100 mg    of    liver,    gastrocnemius    muscle    and    retroperitoneal    adipose    tissue    were    used    for    the    determination    of    TNF-α,    IL-6,    and    IL-10    content.    Tissues    were    homogenized    in    RIPA    buffer    (0.625%    Nonidet    P-40,    0.625%    sodium    deoxycholate,    6.25 mM    sodium    phosphate,    and    1 mM    ethylenediamine    tetra    acetic    acid    at    pH    7.4),    containing    10 g/mL    of    a    protease    inhibitor    cocktail    (Sigma-Aldrich,    St.    Louis,    MO,    USA).    Homogenates    were    centrifuged    at    12,000 g    for    10 min    at    4°C,    supernatant    was    collected,    and    protein    concentration    determined    using    Bradford    assay    (Bio-Rad,    Hercules,    CA,    USA).    Bovine    serum    albumin    was    used    as    a    protein    reference.    Likewise,    about    150 mg    of    epididymal    adipose    tissue    were    cultured    in    DMEM    sterile    medium    (Gibco),    containing    10%    FBS,      2 mM    glutamine    and    antibiotics    for    24 h,    at    37°C,    5%    CO2    humidified    air    environment.    Thereafter,    medium    culture    was    collected    and    used    for    the    determination    of    leptin,    adiponectin,    and    resistin    contents.    For    all    determinations,    we    used    colorimetric    immunoassays    ELISA    (DuoSet    kits,    R&D    System).


#### 2.6.2. Peritoneal    Macrophage    Isolation    and    Culture

Cytokine    and    nitric    oxide    (NO)    production    was    evaluated    in    macrophages    obtained    by    washing    the    peritoneal    cavity    with    6 mL    RPMI    culture    medium    (Gibco),    containing    10%    FBS    and    4 mM    glutamine.    Macrophage-rich    cultures    (more    than    90%    of    the    cells    were    F4/80+)    were    obtained    by    incubating    peritoneal    cells    in    24-well    polystyrene    culture    plates    for    2 h    at    37°C    in    a    5%    CO2,    humidified    air    environment.    Nonadherent    cells    were    removed    by    washing    with    RPMI.    Adherent    cells    were    then    incubated    with    2.5 μg/mL    of    LPS    (E.    coli,    serotype    0111:B4,    Sigma    Chemical    Company,    USA)    for    24 h    [23]. Medium    was    collected    for    determination    of    IL-6,    IL-10,    and    TNF-α    by    ELISA,    and    nitrite    content    by    Griess    method    [24].


### 2.7. Statistical    Analysis

Data    are    presented    as    mean ± SEM.    All    groups    were    compared    by    two-way    ANOVA    following    Bonferroni    posttests.P<0.05    was    considered    to    be    significant.


## 2.1. Animals

All    animal    studies    were    performed    according    to    protocols    approved    by    the    Animal    Care    and    Use    Committee    from    the    Institute    of    Biomedical    Sciences,    University    of    São    Paulo.    C57BL/6    male    mice    (8    weeks-old)    were    used    for    the    study.    Animals    were    housed    in    a    room    with    light-dark    cycle    of    12-12 h    and    temperature    of23±2°C.    Animals    were    divided    in    four    groups:    (a)    control    diet    (CD),    (b)    high-fat    diet    (HFD),    (c)    control    diet    supplemented    with    n-6    (CD    +    n-6),    and    (d)    high-fat    diet    supplemented    with    n-6    (HFD    +    n-6).    During    the    first    4    weeks    preceding    the    induction    of    obesity    by    HFD,    all    the    four    experimental    groups    were    fed    ad    libitum    with    a    control    diet    (76%    carbohydrates,    9%    fat,    15%    proteins).    CD    +    n-6    and    HFD    +    n-6    were    supplemented    with    sunflower    oil    (n-6    PUFA    source)    by    oral    gavage    at    2 g    per    Kg    of    body    weight,    three    times    per    week,    during    12    weeks.    This    dosage    of    oil    was    chosen    based    on    previous    studies    using    different    oils    from    our    group    and    others    [11–13].    CD    and    HFD    received    water    at    the    same    dose.


## 2.2. HFD-Induced    Obesity    and    Insulin    Resistance

After    the    first    4    weeks,    animals    from    the    HFD    and    HFD    +    n-6    groups    received    high-fat    diet    (26%    carbohydrates,    59%    fat,    15%    proteins)    during    the    next    8    weeks.    CD    and    CD    +    n-6    groups    remained    on    the    control    diet.    Supplementation    with    n-6    (sunflower    oil,    2 g/Kg    b.w.)    was    kept    until    the    end    of    the    12    weeks.


## 2.3. Glucose    and    Insulin    Tolerance    Tests

Tolerances    to    glucose    (GTT)    and    to    insulin    (ITT)    were    evaluated    after    6 h    fasting.    For    GTT,    mice    were    intraperitoneally    injected    (i.p.)    with    glucose    (2 g/Kg    body    weight).    Blood    glucose    measurements    were    performed    at    0,    15,    30,    45,    60,    and    90 min    after    glucose    injection.    Glucose    concentration    versus    time    was    plotted    and    the    area    under    the    curve    (AUC)    was    calculated    for    each    animal.    For    ITT,    animals    were    i.p.    injected    with    insulin    (Humulin    R,    Lilly,    0.75 UI/kg    b.w.)    and    glucose    measurements    were    performed    at    0,    10,    20,    30,    40,    50,    and    60 min    after    injection.    Glucose    concentration    versus    time    was    plotted    and    the    glucose    lowering    rate    was    calculated.In    both    tests,    blood    samples    were    collected    from    the    tail    vein.    For    GTT,    serum    glucose    was    measured    by    colorimetric    assay    commercially    available    (PAP    Liquiform    Glucose,    Labtest)    and    for    ITT,    glucose    was    measured    by        using    glucometer    (One    Touch    Ultra,    Johnson    &    Johnson).


## 2.4. Serum    Parameters    Analysis

After    6    hours    fasting,    animals    were    anesthetized    and    blood    was    collected    by    puncturing    the    orbital    plexus.    Serum    glucose,    triacylglycerol,    total    cholesterol,    LDL-cholesterol,    and    HDL-cholesterol    were    determined    by    colorimetric    assays    (Labtest    Diagnostics,    Lagoa    Santa,    MG,    Brazil).


## 2.5. Responsiveness    to    Insulin    in    Isolated    Soleus    Muscle

Animals    were    euthanized    by    cervical    dislocation    and    soleus    muscles    rapidly    and    carefully    isolated,    weighed    (8–10 mg),    attached    to    stainless    steel    clips    to    maintain    resting    tension,    and    preincubated    for    30 min,    at    37°C,    in    Krebs-Ringer    bicarbonate    buffer    (KRBB)    containing    5.6 mM    glucose    and    1%    bovine    serum    albumin    (BSA),    pH    7.4,    pregassed    for    30 min    with    95%    O2/5%    CO2,    with    agitation    at    100    oscillations    per    min.    After    this    period,    muscles    were    transferred    to    other    vials    containing    the    same    buffer,    but    added    of    0.3 μCi/mL    D-[U-14C]-glucose    and    0.2 μCi/mL    2-deoxy-D-[2,6-3H]-glucose.    Phenylethylamine    (0.2 mL),    diluted    1 : 1 v/v    in    methanol,    was    added    into    a    separate    compartment    for    14CO2    adsorption.    Incubation    was    then    performed    for    1 h    under    similar    conditions,    in    the    absence    or    presence    of    7 nM    insulin.    Gasification    with    95%    O2/5%    CO2    was    maintained    during    the    pre-incubation    and    the    first    15 min    of    the    incubation    period    and    then    stopped.After    the    incubation    period,    muscles    were    briefly    washed    in    cold    KRBB    at    4°C,    dried    on    filter    paper    and    frozen    in    liquid    N2.    Samples    were    processed    for    measurements    of    uptake    of    2-deoxy-D-[2,6-3H]-glucose,    D-[14C]-glucose    incorporation,    [14C]-glycogen    synthesis,    and    decarboxylation    of    D-[14C]-glucose,    according    to    methods    described    by    Challiss    et    al.    [14],    Espinal    et    al.    [15]    and    Leighton    et    al.    [16],    respectively,    and    routinely    have    been    used    by    our    group    [11, 17–22].


## 2.6. Analysis    of    Inflammatory    Parameters

### 2.6.1. Tissue    Cytokine    and    Adipokine    Content    Measurements

Mice    were    euthanized    on    CO2    chamber    and    visceral    adipose    tissue    (epididymal,    retroperitoneal,    and    mesenteric),    liver,    gastrocnemius    muscle,    and    peritoneal    macrophages    rapidly    collected.    About    100 mg    of    liver,    gastrocnemius    muscle    and    retroperitoneal    adipose    tissue    were    used    for    the    determination    of    TNF-α,    IL-6,    and    IL-10    content.    Tissues    were    homogenized    in    RIPA    buffer    (0.625%    Nonidet    P-40,    0.625%    sodium    deoxycholate,    6.25 mM    sodium    phosphate,    and    1 mM    ethylenediamine    tetra    acetic    acid    at    pH    7.4),    containing    10 g/mL    of    a    protease    inhibitor    cocktail    (Sigma-Aldrich,    St.    Louis,    MO,    USA).    Homogenates    were    centrifuged    at    12,000 g    for    10 min    at    4°C,    supernatant    was    collected,    and    protein    concentration    determined    using    Bradford    assay    (Bio-Rad,    Hercules,    CA,    USA).    Bovine    serum    albumin    was    used    as    a    protein    reference.    Likewise,    about    150 mg    of    epididymal    adipose    tissue    were    cultured    in    DMEM    sterile    medium    (Gibco),    containing    10%    FBS,      2 mM    glutamine    and    antibiotics    for    24 h,    at    37°C,    5%    CO2    humidified    air    environment.    Thereafter,    medium    culture    was    collected    and    used    for    the    determination    of    leptin,    adiponectin,    and    resistin    contents.    For    all    determinations,    we    used    colorimetric    immunoassays    ELISA    (DuoSet    kits,    R&D    System).


### 2.6.2. Peritoneal    Macrophage    Isolation    and    Culture

Cytokine    and    nitric    oxide    (NO)    production    was    evaluated    in    macrophages    obtained    by    washing    the    peritoneal    cavity    with    6 mL    RPMI    culture    medium    (Gibco),    containing    10%    FBS    and    4 mM    glutamine.    Macrophage-rich    cultures    (more    than    90%    of    the    cells    were    F4/80+)    were    obtained    by    incubating    peritoneal    cells    in    24-well    polystyrene    culture    plates    for    2 h    at    37°C    in    a    5%    CO2,    humidified    air    environment.    Nonadherent    cells    were    removed    by    washing    with    RPMI.    Adherent    cells    were    then    incubated    with    2.5 μg/mL    of    LPS    (E.    coli,    serotype    0111:B4,    Sigma    Chemical    Company,    USA)    for    24 h    [23]. Medium    was    collected    for    determination    of    IL-6,    IL-10,    and    TNF-α    by    ELISA,    and    nitrite    content    by    Griess    method    [24].


## 2.6.1. Tissue    Cytokine    and    Adipokine    Content    Measurements

Mice    were    euthanized    on    CO2    chamber    and    visceral    adipose    tissue    (epididymal,    retroperitoneal,    and    mesenteric),    liver,    gastrocnemius    muscle,    and    peritoneal    macrophages    rapidly    collected.    About    100 mg    of    liver,    gastrocnemius    muscle    and    retroperitoneal    adipose    tissue    were    used    for    the    determination    of    TNF-α,    IL-6,    and    IL-10    content.    Tissues    were    homogenized    in    RIPA    buffer    (0.625%    Nonidet    P-40,    0.625%    sodium    deoxycholate,    6.25 mM    sodium    phosphate,    and    1 mM    ethylenediamine    tetra    acetic    acid    at    pH    7.4),    containing    10 g/mL    of    a    protease    inhibitor    cocktail    (Sigma-Aldrich,    St.    Louis,    MO,    USA).    Homogenates    were    centrifuged    at    12,000 g    for    10 min    at    4°C,    supernatant    was    collected,    and    protein    concentration    determined    using    Bradford    assay    (Bio-Rad,    Hercules,    CA,    USA).    Bovine    serum    albumin    was    used    as    a    protein    reference.    Likewise,    about    150 mg    of    epididymal    adipose    tissue    were    cultured    in    DMEM    sterile    medium    (Gibco),    containing    10%    FBS,      2 mM    glutamine    and    antibiotics    for    24 h,    at    37°C,    5%    CO2    humidified    air    environment.    Thereafter,    medium    culture    was    collected    and    used    for    the    determination    of    leptin,    adiponectin,    and    resistin    contents.    For    all    determinations,    we    used    colorimetric    immunoassays    ELISA    (DuoSet    kits,    R&D    System).


## 2.6.2. Peritoneal    Macrophage    Isolation    and    Culture

Cytokine    and    nitric    oxide    (NO)    production    was    evaluated    in    macrophages    obtained    by    washing    the    peritoneal    cavity    with    6 mL    RPMI    culture    medium    (Gibco),    containing    10%    FBS    and    4 mM    glutamine.    Macrophage-rich    cultures    (more    than    90%    of    the    cells    were    F4/80+)    were    obtained    by    incubating    peritoneal    cells    in    24-well    polystyrene    culture    plates    for    2 h    at    37°C    in    a    5%    CO2,    humidified    air    environment.    Nonadherent    cells    were    removed    by    washing    with    RPMI.    Adherent    cells    were    then    incubated    with    2.5 μg/mL    of    LPS    (E.    coli,    serotype    0111:B4,    Sigma    Chemical    Company,    USA)    for    24 h    [23]. Medium    was    collected    for    determination    of    IL-6,    IL-10,    and    TNF-α    by    ELISA,    and    nitrite    content    by    Griess    method    [24].


## 2.7. Statistical    Analysis

Data    are    presented    as    mean ± SEM.    All    groups    were    compared    by    two-way    ANOVA    following    Bonferroni    posttests.P<0.05    was    considered    to    be    significant.


## 3. Results

### 3.1. Exposure    to    HFD    Induces    Obesity    Associated    with    Glucose    and    Insulin    Intolerance

Animals    fed    with    HFD    for    eight    weeks    showed    increased    (by    3.8    fold)    body    weight    gain    when    compared    to    those    fed    with    CD.    Despite    reduced    food    ingestion,    the    food    efficiency    of    HFD    was    6    fold    higher    than    CD.    Moreover,    epididymal    adipose    tissues    were    increased    by    HFD.    Sunflower    oil    did    not    change    body    weight    gain,    food    efficiency,    or    adipose    tissue    increased    (Table1).Table 1
Obesity characteristics from mice fed with control diet (CD) or high-fat diet (HFD), supplemented or not with sunflower oil (rich in n-6 fatty acids).
CD
HFD
CD + n6
HFD + n6
Body weight gain (g)
2.90 ± 0.42
11.30
±
0.91
*
3.99 ± 0.65
10.33 ± 0.50#
Food ingestion (g/day/animal)
3.61 ± 0.091
2.44
±
0.164
*
4.53
±
0.158
*
2.45 ± 0.033#
Food efficiency (body weight gain (g)/food ingestion (g))
0.014 ± 0.06
0.083
±
0.022
*
0.016 ± 0.008
0.075 ± 0.011#
Epididymal adipose tissue (mg)
737.9 ± 89.4
1777.0
±
234.3
*
555.1 ± 45.5
1450.0 ± 176.8#
Oral supplementation with sunflower oil rich in n-6 (2 g/Kg body weight, three times a week, oral gavage) or water, started four weeks before feeding or not with HFD, maintained until the end of experimental protocol. Animals were feed with HFD or CD for additional eight weeks. Mean ± SEM (n = 8–10). Data were analyzed by two-way ANOVA and Bonferroni post-test. *P<0.05 versus CD; #P<0.05 versus CD + n6.HFD    increased    fasted    glucose    in    18%    (Table2).    An    increase    of    63%    in    area    under    the    curve    in    glucose    tolerance    test    (GTT)    was    also    observed    in    animals    treated    with    HDF,    demonstrating    glucose    intolerance,    which    was    associated    with    insulin    intolerance,    as    observed    by    the    reduction    in    glucose    clearance    during    insulin    tolerance    test    (ITT).    Supplementation    with    sunflower    oil    maintained    fasted    glucose    levels    similar    to    CD.    However,    the    supplementation    did    not    prevent    the    glucose    and    insulin    intolerance    induced    by    HFD.    Furthermore,    sunflower    oil    supplementation    induced    insulin    intolerance    itself    (CD    +    n-6    group),    reducing    the    clearance    of    glucose    similar    to    HFD    without    supplementation.    Total    cholesterol    and    LDL    cholesterol    levels    were    increased    by    HFD    and    sunflower    oil    supplementation    in    association    with    HFD    showed    protective    effect    in    the    prevention    of    the    increased    LDL    cholesterol    and    cholesterol    total    levels    found    in    HFD.Table 2
Metabolic parameters from mice fed with control diet (CD) or high-fat diet (HFD), supplemented, or not with sunflower oil (rich in n-6 fatty acids).
CD
HFD
CD + n6
HFD + n6
Fasted glucose (mg/dL)
186.1 ± 5.66
221.0
±
8.38
*
173.8 ± 4.75
196.8 ± 10.66
GTT test (AUC)
7987 ± 779.3
13090
±
643.9
*
7568 ± 1359
14790 ± 1731#
ITT test (% glucose/min)
2.97 ± 0.19
2.13
±
0.21
*
1.94
±
0.30
*
1.98 ± 0.22
Total cholesterol (mg/dL)
148.2 ± 6.72
192.7
±
11.96
*
164.3 ± 14.00
166.3 ± 12.33
LDL cholesterol (mg/dL)
127.4 ± 5.15
165.1
±
14.49
*
128.2 ± 13.77
136.7 ± 13.22
HDL cholesterol (mg/dL)
14.73 ± 0.83
15.52 ± 1.86
19.33 ± 2.21
15.00 ± 1.15
Triglycerides (mg/dL)
68.61 ± 5.56
63.00 ± 4.40
84.70 ± 8.53
72.78 ± 8.71
Oral supplementation with n-6 (2 g/Kg body weight, three times a week, gavage) or water have started four weeks before HFD and continued until the end of experimental period. Animals were fed HFD or CD for eight weeks. GTT: glucose tolerance test; ITT: insulin tolerance test. Mean ± SEM (n = 6–15). ANOVA two-way with Bonferroni post tests. *P<0.05 when compared with CD; #P<0.05 when compared with CD + n6.


### 3.2. HFD    Impaired    Glucose    Uptake    and    Metabolism    in    Soleus    Muscle

Soleus    muscles    from    animals    fed    with    HFD    did    not    respond    to    insulin    stimulus    in    relation    to    glucose    uptake    and    metabolism    (Figure1),    characterizing    muscle    insulin    resistance    state.    This    effect    was    not    prevented    or    reduced    by    sunflower    oil    supplementation.    Interestingly,    the    supplementation    itself    (CD    +    n-6    group)    increased    basal    glucose    uptake    and    glucose    oxidation,    but    it    failed    to    respond    to    the    insulin    stimulus    when    compared    to    the    control    group    (CD    group).Effect    of    sunflower    oil    supplementation    (rich    in    n-6    fatty    acids)    on    glucose    uptake    and    metabolism    in    isolated    soleus    muscle    from    C57BL/6    mice.    The    muscles    were        incubated    in    Krebs    Ringer    buffer,    containing    5.6 mM    glucose,    0.3μCi/mL    of    [U-14C]-D-glucose    and    0.2 μCi/mL    of    2-deoxy-[2,6-3H]-D-glucose,    in    the    absence    (white    bars)    or    presence    (black    bars)    of    7 nM    insulin,    for    one    hour.    At    the    end    of    the    incubation    period,    muscles    were    proceeded    to    analyze    glucose    uptake    and    metabolism    as    described    in    Materials    and    Methods.    Results    are    presented    as    mean    ±    SEM    (n = 10–16).    Two-way    ANOVA    and    Bonferroni    posttest    were    used    for    statistical    analysis.    §P<0.05    versus    the    insulin    stimulated    (+insulin)    of    the    same    group;    *P<0.05    versus    CD    with    insulin;    #P<0.05    versus    CD    +    n6    with    insulin.
(a)
(b)
(c)
(d)


### 3.3. HFD    and    Sunflower    Oil    Supplementation    Induced    Inflammation

Peritoneal    macrophages    stimulated    with    LPS    showed    increased    production    of    nitrite,    TNFα,    IL6,    and    IL10    when    compared    with    unstimulated    cells    (Figure 2).    Macrophages    from    animals    with    obesity    induced    by    HFD    for    8    weeks    showed    a    inflammatory    profile,    with    increased    production    of    nitrite,    TNF-α,    and    IL-6,    associated    with    decreased    content    of    IL-10    when    stimulated    with    LPS.    Interestingly,    supplementation    with    sunflower    oil    induced    similar    inflammatory    response    in    macrophages.    For    TNF-α    only,    the    association    of    HFD    and    n-6    supplementation    (HFD    +    n-6    group)    showed    beneficial    response,    decreasing    this    cytokine    production.Nitric    oxide    and    cytokine    production    in    peritoneal    macrophages    collected    from    mice    fed    with    control    diet    (CD)    or    high-fat    diet    (HFD),    supplemented    or    not    with    sunflower    oil    (rich    in    n-6    fatty    acids,    2 g/Kg    b.w.,    three    times    a    week,    by    oral    gavage).    Animals    were    fed    with    HFD    or    CD    for    eight    weeks.    Peritoneal    macrophages    were    collected    and    cultured    for    24 h    in    the    absence    (white    bars)    or    presence    (black    bars)    of    2.5μg/mL    LPS.    Nitric    oxide    (a),    TNF-α    (b),    IL-6    (c),    and    IL-10    (d)    were    measured    as    described    in    Material    and    Methods.    Total    number    of    cells    in    peritoneal    exudates    was    not    different    among    the    groups    (data    not    shown).    Data    are    presented    as    mean    ±    SEM    (n=5–10).    The    CD    +    n6    group    had    just    5    samples    up    to    the    detection    limit    in    the    TNF-α    assay.    Two-way    ANOVA    and    Bonferroni    post-test    were    used    for    statistical    analysis.    All    LPS    stimulated    conditions    were    different    from    unstimulated;    *P<0.05    versus    CD    with    LPS;    #P<0.05    versus    CD    +    n6    with    LPS;    °P<0.05    versus    HFD    with    LPS.
(a)
(b)
(c)
(d)In    general,    HFD    induced    and    increase    in    the    pro-inflammatory    cytokine    IL6    and    decrease    in    the    anti-inflammatory    cytokine    IL10    contents    in    the    insulin    target    tissues    (liver,    skeletal    muscle,    and    adipose    tissue;    Figure3).    Interesting,    sunflower    oil    supplementation    itself    (CD    +    n-6    group)    for    12    weeks    increased    the    content    of    the    proinflammatory    cytokine    IL6    in    all    tissues.    However,    when    the    sunflower    oil    was    associated    with    HFD    (HFD    +    n-6    group),    cytokine    concentrations    were    similar    to    that    found    in    the    HFD    group,    in    liver    and    adipose    tissue.Tissue    cytokine    concentrations    of    TNFα    (a),    IL6    (b),    and    IL10    (c)    in    adipose    tissue    (retroperitoneal),    liver,    and    gastrocnemius    skeletal    muscle    from    mice    treated    with    control    diet    (CD)    or    high-fat    diet    (HFD),    supplemented    or    not    with    sunflower    oil    (rich    in    n-6    fatty    acids).    Oral    supplementation    with    n-6    (2 g/Kg    body    weight,    three    times    a    week,    gavage)    or    water    started    four    weeks    before    HFD    and    continued    until    the    end    of    experiment.    Animals    were    feed    with    HFD    or    CD    for    eight    weeks.    Cytokines    were    measured    in    tissue    homogenate.    Values    are    expressed    as    pg/mg    protein.    Mean    ±    SEM    (n=4–6).    The    statistical    analysis    was    performed    using    two-way    ANOVA    with    Bonferroni    post    tests.    *P<0.05    when    compared    with    CD;    #P<0.05    when    compared    with    CD    +    n6.
(a)
(b)
(c)Adipokines    production    by    epididymal    adipose    tissue    was    determined    after    24 h    culture.    Adipose    tissues    from    HFD    animals    showed    increased    production    of    leptin    (Table3).    Resistin    was    reduced    only    when    HFD    was    associated    to    sunflower    oil    supplementation    (HFD    +    n-6    group)    in    comparison    to    CD    +    n6    group.Table 3
Adipokine production in adipose tissue from mice fed with control diet (CD) or high-fat diet (HFD), supplemented, or not with sunflower oil (rich in n-6 fatty acids).
CD
HFD
CD + n6
HFD + n6
Epididymal adipose tissuea
Leptin
42.51 ± 8.75
81.38
±
9.90
*
62.42 ± 8.80
88.03 ± 10.96
Adiponectin
659.9 ± 39.94
697.6 ± 43.85
730.3 ± 76.68
649.8 ± 45.38
Resistin
625.1 ± 100.8
528.6 ± 71.65
913.0 ± 181.0
327.0 ± 42.15#
Oral supplementation with n-6 (2 g/Kg body weight, three times a week, gavage) or water started four weeks before HFD and continued until the end of experiment. Animals were feed with HFD or CD for eight weeks. Approximately 150 mg of tissue were incubated in DMEM medium for 24 h and the supernatant were used for measures. Mean ± SEM (n = 7–11). ANOVA two-way with Bonferroni post tests. *P<0.05 when compared with CD; #P<0.05 when compared with CD + n6. aValues are expressed as pg/mL/mg tissue.


## 3.1. Exposure    to    HFD    Induces    Obesity    Associated    with    Glucose    and    Insulin    Intolerance

Animals    fed    with    HFD    for    eight    weeks    showed    increased    (by    3.8    fold)    body    weight    gain    when    compared    to    those    fed    with    CD.    Despite    reduced    food    ingestion,    the    food    efficiency    of    HFD    was    6    fold    higher    than    CD.    Moreover,    epididymal    adipose    tissues    were    increased    by    HFD.    Sunflower    oil    did    not    change    body    weight    gain,    food    efficiency,    or    adipose    tissue    increased    (Table1).Table 1
Obesity characteristics from mice fed with control diet (CD) or high-fat diet (HFD), supplemented or not with sunflower oil (rich in n-6 fatty acids).
CD
HFD
CD + n6
HFD + n6
Body weight gain (g)
2.90 ± 0.42
11.30
±
0.91
*
3.99 ± 0.65
10.33 ± 0.50#
Food ingestion (g/day/animal)
3.61 ± 0.091
2.44
±
0.164
*
4.53
±
0.158
*
2.45 ± 0.033#
Food efficiency (body weight gain (g)/food ingestion (g))
0.014 ± 0.06
0.083
±
0.022
*
0.016 ± 0.008
0.075 ± 0.011#
Epididymal adipose tissue (mg)
737.9 ± 89.4
1777.0
±
234.3
*
555.1 ± 45.5
1450.0 ± 176.8#
Oral supplementation with sunflower oil rich in n-6 (2 g/Kg body weight, three times a week, oral gavage) or water, started four weeks before feeding or not with HFD, maintained until the end of experimental protocol. Animals were feed with HFD or CD for additional eight weeks. Mean ± SEM (n = 8–10). Data were analyzed by two-way ANOVA and Bonferroni post-test. *P<0.05 versus CD; #P<0.05 versus CD + n6.HFD    increased    fasted    glucose    in    18%    (Table2).    An    increase    of    63%    in    area    under    the    curve    in    glucose    tolerance    test    (GTT)    was    also    observed    in    animals    treated    with    HDF,    demonstrating    glucose    intolerance,    which    was    associated    with    insulin    intolerance,    as    observed    by    the    reduction    in    glucose    clearance    during    insulin    tolerance    test    (ITT).    Supplementation    with    sunflower    oil    maintained    fasted    glucose    levels    similar    to    CD.    However,    the    supplementation    did    not    prevent    the    glucose    and    insulin    intolerance    induced    by    HFD.    Furthermore,    sunflower    oil    supplementation    induced    insulin    intolerance    itself    (CD    +    n-6    group),    reducing    the    clearance    of    glucose    similar    to    HFD    without    supplementation.    Total    cholesterol    and    LDL    cholesterol    levels    were    increased    by    HFD    and    sunflower    oil    supplementation    in    association    with    HFD    showed    protective    effect    in    the    prevention    of    the    increased    LDL    cholesterol    and    cholesterol    total    levels    found    in    HFD.Table 2
Metabolic parameters from mice fed with control diet (CD) or high-fat diet (HFD), supplemented, or not with sunflower oil (rich in n-6 fatty acids).
CD
HFD
CD + n6
HFD + n6
Fasted glucose (mg/dL)
186.1 ± 5.66
221.0
±
8.38
*
173.8 ± 4.75
196.8 ± 10.66
GTT test (AUC)
7987 ± 779.3
13090
±
643.9
*
7568 ± 1359
14790 ± 1731#
ITT test (% glucose/min)
2.97 ± 0.19
2.13
±
0.21
*
1.94
±
0.30
*
1.98 ± 0.22
Total cholesterol (mg/dL)
148.2 ± 6.72
192.7
±
11.96
*
164.3 ± 14.00
166.3 ± 12.33
LDL cholesterol (mg/dL)
127.4 ± 5.15
165.1
±
14.49
*
128.2 ± 13.77
136.7 ± 13.22
HDL cholesterol (mg/dL)
14.73 ± 0.83
15.52 ± 1.86
19.33 ± 2.21
15.00 ± 1.15
Triglycerides (mg/dL)
68.61 ± 5.56
63.00 ± 4.40
84.70 ± 8.53
72.78 ± 8.71
Oral supplementation with n-6 (2 g/Kg body weight, three times a week, gavage) or water have started four weeks before HFD and continued until the end of experimental period. Animals were fed HFD or CD for eight weeks. GTT: glucose tolerance test; ITT: insulin tolerance test. Mean ± SEM (n = 6–15). ANOVA two-way with Bonferroni post tests. *P<0.05 when compared with CD; #P<0.05 when compared with CD + n6.


## 3.2. HFD    Impaired    Glucose    Uptake    and    Metabolism    in    Soleus    Muscle

Soleus    muscles    from    animals    fed    with    HFD    did    not    respond    to    insulin    stimulus    in    relation    to    glucose    uptake    and    metabolism    (Figure1),    characterizing    muscle    insulin    resistance    state.    This    effect    was    not    prevented    or    reduced    by    sunflower    oil    supplementation.    Interestingly,    the    supplementation    itself    (CD    +    n-6    group)    increased    basal    glucose    uptake    and    glucose    oxidation,    but    it    failed    to    respond    to    the    insulin    stimulus    when    compared    to    the    control    group    (CD    group).Effect    of    sunflower    oil    supplementation    (rich    in    n-6    fatty    acids)    on    glucose    uptake    and    metabolism    in    isolated    soleus    muscle    from    C57BL/6    mice.    The    muscles    were        incubated    in    Krebs    Ringer    buffer,    containing    5.6 mM    glucose,    0.3μCi/mL    of    [U-14C]-D-glucose    and    0.2 μCi/mL    of    2-deoxy-[2,6-3H]-D-glucose,    in    the    absence    (white    bars)    or    presence    (black    bars)    of    7 nM    insulin,    for    one    hour.    At    the    end    of    the    incubation    period,    muscles    were    proceeded    to    analyze    glucose    uptake    and    metabolism    as    described    in    Materials    and    Methods.    Results    are    presented    as    mean    ±    SEM    (n = 10–16).    Two-way    ANOVA    and    Bonferroni    posttest    were    used    for    statistical    analysis.    §P<0.05    versus    the    insulin    stimulated    (+insulin)    of    the    same    group;    *P<0.05    versus    CD    with    insulin;    #P<0.05    versus    CD    +    n6    with    insulin.
(a)
(b)
(c)
(d)


## 3.3. HFD    and    Sunflower    Oil    Supplementation    Induced    Inflammation

Peritoneal    macrophages    stimulated    with    LPS    showed    increased    production    of    nitrite,    TNFα,    IL6,    and    IL10    when    compared    with    unstimulated    cells    (Figure 2).    Macrophages    from    animals    with    obesity    induced    by    HFD    for    8    weeks    showed    a    inflammatory    profile,    with    increased    production    of    nitrite,    TNF-α,    and    IL-6,    associated    with    decreased    content    of    IL-10    when    stimulated    with    LPS.    Interestingly,    supplementation    with    sunflower    oil    induced    similar    inflammatory    response    in    macrophages.    For    TNF-α    only,    the    association    of    HFD    and    n-6    supplementation    (HFD    +    n-6    group)    showed    beneficial    response,    decreasing    this    cytokine    production.Nitric    oxide    and    cytokine    production    in    peritoneal    macrophages    collected    from    mice    fed    with    control    diet    (CD)    or    high-fat    diet    (HFD),    supplemented    or    not    with    sunflower    oil    (rich    in    n-6    fatty    acids,    2 g/Kg    b.w.,    three    times    a    week,    by    oral    gavage).    Animals    were    fed    with    HFD    or    CD    for    eight    weeks.    Peritoneal    macrophages    were    collected    and    cultured    for    24 h    in    the    absence    (white    bars)    or    presence    (black    bars)    of    2.5μg/mL    LPS.    Nitric    oxide    (a),    TNF-α    (b),    IL-6    (c),    and    IL-10    (d)    were    measured    as    described    in    Material    and    Methods.    Total    number    of    cells    in    peritoneal    exudates    was    not    different    among    the    groups    (data    not    shown).    Data    are    presented    as    mean    ±    SEM    (n=5–10).    The    CD    +    n6    group    had    just    5    samples    up    to    the    detection    limit    in    the    TNF-α    assay.    Two-way    ANOVA    and    Bonferroni    post-test    were    used    for    statistical    analysis.    All    LPS    stimulated    conditions    were    different    from    unstimulated;    *P<0.05    versus    CD    with    LPS;    #P<0.05    versus    CD    +    n6    with    LPS;    °P<0.05    versus    HFD    with    LPS.
(a)
(b)
(c)
(d)In    general,    HFD    induced    and    increase    in    the    pro-inflammatory    cytokine    IL6    and    decrease    in    the    anti-inflammatory    cytokine    IL10    contents    in    the    insulin    target    tissues    (liver,    skeletal    muscle,    and    adipose    tissue;    Figure3).    Interesting,    sunflower    oil    supplementation    itself    (CD    +    n-6    group)    for    12    weeks    increased    the    content    of    the    proinflammatory    cytokine    IL6    in    all    tissues.    However,    when    the    sunflower    oil    was    associated    with    HFD    (HFD    +    n-6    group),    cytokine    concentrations    were    similar    to    that    found    in    the    HFD    group,    in    liver    and    adipose    tissue.Tissue    cytokine    concentrations    of    TNFα    (a),    IL6    (b),    and    IL10    (c)    in    adipose    tissue    (retroperitoneal),    liver,    and    gastrocnemius    skeletal    muscle    from    mice    treated    with    control    diet    (CD)    or    high-fat    diet    (HFD),    supplemented    or    not    with    sunflower    oil    (rich    in    n-6    fatty    acids).    Oral    supplementation    with    n-6    (2 g/Kg    body    weight,    three    times    a    week,    gavage)    or    water    started    four    weeks    before    HFD    and    continued    until    the    end    of    experiment.    Animals    were    feed    with    HFD    or    CD    for    eight    weeks.    Cytokines    were    measured    in    tissue    homogenate.    Values    are    expressed    as    pg/mg    protein.    Mean    ±    SEM    (n=4–6).    The    statistical    analysis    was    performed    using    two-way    ANOVA    with    Bonferroni    post    tests.    *P<0.05    when    compared    with    CD;    #P<0.05    when    compared    with    CD    +    n6.
(a)
(b)
(c)Adipokines    production    by    epididymal    adipose    tissue    was    determined    after    24 h    culture.    Adipose    tissues    from    HFD    animals    showed    increased    production    of    leptin    (Table3).    Resistin    was    reduced    only    when    HFD    was    associated    to    sunflower    oil    supplementation    (HFD    +    n-6    group)    in    comparison    to    CD    +    n6    group.Table 3
Adipokine production in adipose tissue from mice fed with control diet (CD) or high-fat diet (HFD), supplemented, or not with sunflower oil (rich in n-6 fatty acids).
CD
HFD
CD + n6
HFD + n6
Epididymal adipose tissuea
Leptin
42.51 ± 8.75
81.38
±
9.90
*
62.42 ± 8.80
88.03 ± 10.96
Adiponectin
659.9 ± 39.94
697.6 ± 43.85
730.3 ± 76.68
649.8 ± 45.38
Resistin
625.1 ± 100.8
528.6 ± 71.65
913.0 ± 181.0
327.0 ± 42.15#
Oral supplementation with n-6 (2 g/Kg body weight, three times a week, gavage) or water started four weeks before HFD and continued until the end of experiment. Animals were feed with HFD or CD for eight weeks. Approximately 150 mg of tissue were incubated in DMEM medium for 24 h and the supernatant were used for measures. Mean ± SEM (n = 7–11). ANOVA two-way with Bonferroni post tests. *P<0.05 when compared with CD; #P<0.05 when compared with CD + n6. aValues are expressed as pg/mL/mg tissue.


## 4. Discussion

This    is    the    first    study    to    investigate    the    effects    of    sunflower    oil    supplementation    on    inflammation    and    insulin    sensitivity    in    mice    fed    a    balanced    diet    and    a    HFD.    We    showed    that    HFD    induces    decreased    glucose    tolerance    (as    demonstrated    by    GTT)    and    insulin    sensitivity    (as    observed    in    ITT)    in    the    whole    body.    Surprisingly,    this    last    effect    was    also    verified    in    the    CD    +    n6    group.    Moreover,    proinflammatory    response    was    increased    in    the    HFD    group    without    prevention    or    attenuation    by    sunflower    oil    supplementation.    This    effect    was    also    observed    in    CD    +    n6    group.    Lipid    profile    was    impaired    by    HFD.    Animals    from    HFD    group    showed    an    increase    in    total    and    LDL    cholesterol    plasma    levels.    These    alterations    were    ameliorated    by    sunflower    oil    supplementation    (HFD    +    n6    group).HFD    has    been    associated    with    insulin    resistance    and    inflammatory    condition,    contributing    to    the    development    of    several    related    diseases,    such    as    obesity,    diabetes    mellitus    type    2,    metabolic    syndrome,    cancer,    and    cardiovascular    diseases    [25–27].    Various    strategies    have    been    investigated    in    order    to    decreasing    insulin    resistance    and    inflammation,    including    natural    and    modified    compounds,    cyclooxygenase    inhibitors,    and    physical    exercise    [28–35].    Since    skeletal    muscle    tissue    represents    50–60%    of    the    body    weight    and    it    is    the    main    insulin-responsive    tissue    in    association    to    liver    and    adipose    tissue,    it    has    been    proposed    that    skeletal    muscle    dysfunctions    (reduced    oxidative    capacity    and    impaired    insulin    sensitivity)    play    a    central    role    in    the    abnormalities    of    chronic    diseases    [21, 36–40].    In    this    work,    we    investigated    the    effect    of    sunflower    oil    supplementation    on    insulin    sensitivity    (in    whole    animal    in    vivo    and    in    soleus    muscle    in    vivo)    and    inflammatory    condition    (liver,    skeletal    muscle,    adipose    tissue)    in    mice    fed    a    balanced    diet    and    a    HFD.    In    recent    study,    Bjermo    et    al.    [41]    showed    that    isocaloric    diet    with    n-6    PUFA    for    10    weeks    reduce    liver    fat    and    modestly    improves    metabolic    conditions,    without    weight    loss    in    humans.    In    addition,    it    has    been    proposed    that    n-6    PUFA    reduce    risks    of    cardiovascular    diseases,    by    improving    lipid    profile    [42].    This    improvement    in    lipid    profile    was    found    in    our    study,    whereas    HFD    +    n6    group    showed    decreased    plasma    LDL    and    total    cholesterol    levels.Some    studies    have    shown    that    n-6    PUFA    intake    positively    correlate    with    obesity    and    insulin    resistance    [43, 44].    Treatment    with    subcutaneous    injection    of    sunflower    oil    by    7    days    in    Wistar    rats    impaired    insulin    sensitivity    by    decreasing    glucose    clearance    during    ITT    and    GLUT-4    expression    and    translocation    in    white    adipose    tissue    (WAT),    with    no    effect    in    skeletal    muscle    [7].    In    the    CD    +    n6    group,    KiTT    was    reduced,    but    glucose    uptake    and    metabolism    in    isolated    soleus    muscles    were    not    altered    after    insulin    stimulus.    These    results    suggest    that    sunflower    oil    supplementation    per    se    is    not    able    to    impair    insulin    response    in    skeletal    muscle,    but    it    possibly    reduces    total    insulin    sensitivity    by    decreasing    the    response    to    the    hormone    in    other    tissues,    such    as    liver    and    adipose    tissue.    This    proposition    has    to    be    investigated    in    further    studies.    In    addition,    both    groups    fed    with    HDF    (HFD    and    HFD    +    n6    groups)    showed    skeletal    muscle    resistance    to    insulin,    showing    no    beneficial    effects    of    n-6    PUFA    on    peripheral    insulin    sensitivity.Obesity    is    accompanied    by    a    chronic    low    grade    subclinical    inflammation    promoted    by    an    increase    in    fuel    availability,    leading    to    infiltration    of    macrophages    into    adipose    tissue.    This    condition    results    in    enhanced    inflammatory    response,    which    stimulates    the    production    of    cytokines    by    adipose    tissue    [45].    Moreover,    in    obesity,    increased    lipotoxicity    caused    by    high    nonesterified    fatty    acids    (NEFA)    available,    and    triacylglycerol    content    leads    to    an    increase    in    inflammatory    markers    and    cytokine    production    in    multiple    organs    [46, 47].    NEFA    increases    inflammatory    response    by    activating    TLR-4    [48].    HFD    increased    inflammatory    response    in    macrophages,    showing    high    content    of    IL-6,    TNF-α,    and    NO,    and    low    content    of    IL-10    (anti-inflammatory    cytokine)    when    compared    to    the    control    group.    In    peripheral    tissues,    IL-6    content    was    increased    in    the    liver    and    gastrocnemius    muscle    whereas    IL-10    content    was    decreased    in    liver    and    retroperitoneal    adipose    tissue.    Adipose    tissue    showed    increased    production    of    leptin    in    the    HFD    and    HFD    +    n6    groups,    compared    with    mice    fed    with    control    diet,    and    resistin    was    reduced    in    the    HFD    +    n6    group    compared    with    CD    +    n6,    in    epididymal    adipose    tissue.    Whereas    in    retroperitoneal    adipose    tissue,    IL-6    was    elevated    in    the    HFD    and    CD    +    n6    groups    compared    with    CD,    IL-10    was    reduced    in    the    HFD    and    CD    +    n6,    and    adiponectin    was    reduced    only    in    CD    +    n6.    These    results    showed    the    inflammatory    characteristics    the    sunflower    oil    supplementation    and    the    high    fatty    diet    fed.Our    results    showed    that    sunflower    oil    supplementation    causes    a    clear    pro-inflammatory    response    in    mice    fed    on    control    diet    and    it    does    not    have    additive    or    prophylactic    effects    in    mice    fed    on    HFD.    Fatty    acid    effects    on    immune    system    have    been    studied    since    1970’s    decade    [49].    Linoleic    acid    is    an    essential    fatty    acid    that    leads    to    inflammatory    response    by    increasing    arachidonic    acid    production,    a    fatty    acid    related    to    elevated    pro-inflammatory    mediator    generation    [50].    High    intake    of    linoleic    acid    has    been    associated    to    the    development    of    chronic    diseases    in    several    studies    [51, 52].    However,    few    studies    have    been    carried    out    on    the    direct    effect    of    this    fatty    acid    on    inflammatory    markers    [53].In    conclusion,    our    results    showed    that    sunflower    oil    supplementation    has    pro-inflammatory    effects    in    macrophages    and    insulin-sensitive    peripheral    tissues    and    induced    insulin    resistancein    vivo    and    in    vitro.    These    effects    were    demonstrated    in    mice    fed    a    balanced    diet    and    pronounced    in    mice    fed    a    HFD.    Interestingly,    sunflower    oil    supplementation    partially    prevented    the    dislipidemia    found    in    mice    submitted    to    HFD,    but    it    did    not    improve    or    even    worsened    inflammatory    state    and    insulin    resistance    induced    by    this    diet.



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*Source: 945131-2012-09-03.xml*