Fig. 1Glucose-stimulated insulin secretion (GSIS) and viability of MIN6 (mouse insulinoma) cells treated with the free fatty acid (FFA) mixtures corresponding to the different profiles. MIN6 cells were treated for 24 (A, C) or 48 hours (B, D) with the FFA mixtures corresponding to the profiles of adolescents of normal weight (NW), obese adolescents without metabolic syndrome (MetS) (ONMS) or obese adolescents with MetS (OWMS). (A, B) Control cells were grown without adding fatty acids. The percentage change in GSIS is shown. (C, D) Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with the results normalized to the control, and expressed as a viability percentage. aIndicates P<0.05, bP<0.01, cP<0.001 using analysis of variance followed by Tukey post hoc test for multiple comparisons. The cells were treated with methanol as negative control for viability (n=3 independent experiments).
Fig. 2Mitochondrial function, lipid peroxidation, and antioxidant capacity of MIN6 (mouse insulinoma) cells treated with free fatty acids (FFAs). MIN6 cells were treated for 24 (A, C, E, G) or 48 (B, D, F, H) hours with the FFA mixtures corresponding to the profiles of adolescents with normal weight (NW), obese adolescents without metabolic syndrome (MetS) (ONMS) or obese adolescents with MetS (OWMS). Control cells were grown without adding fatty acids. (A, B) The potential of the internal mitochondrial membrane was quantified as the mean intensity of fluorescence (MIF) emitted by MitoTracker using flow cytometry analysis. (C, D) The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was determined using bioluminescence. (E, F) The production of lipid peroxidation products was quantified using the thiobarbituric acid reactive substance assay and expressed as the concentration of malondialdehyde (MDA) normalized with the total mg of protein. (G, H) The capacity to trap peroxynitrite is expressed relative to the antioxidant activity of ascorbic acid (AA) with normalization to the total protein content of each group. AU, arbitrary unit. aIndicates P<0.05, bP<0.001 using analysis of variance followed by Tukey test of multiple comparisons.
Fig. 3Effect of the proportion of free fatty acids (FFAs) on the function and viability of MIN6 (mouse insulinoma). The concentration of fatty acids corresponding to the lipid profile of obese adolescents with metabolic syndrome (OWMS) adolescents was adjusted to the total quantity of FFAs present in the serum of normal weight (NW) adolescents (103.25 mg/dL), but the original proportions were conserved (Table 2). MIN6 cells were treated for 24 (A, C) or 48 hours (B, D) with the NW or OWMS profiles. Control cells were grown without adding fatty acids. (A, B) The cells were maintained in baseline conditions of insulin secretion (2 mmol/L glucose in phosphate-buffered saline) and then stimulated with 25 mmol/L glucose in culture medium lacking both FFAs and fetal bovine serum. The percentage change in glucose-stimulated insulin secretion (GSIS) is shown. (C, D) Cellular viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, normalized to the control and expressed as the viability percentage. aIndicates P<0.05, bP<0.01, using analysis of variance followed by Tukey test of multiple comparisons. The cells were treated with methanol as negative control for viability.
Fig. 4Effect of the proportion of free fatty acids (FFAs) on mitochondrial function, lipid peroxidation, and antioxidant capacity of MIN6 (mouse insulinoma) cells. The concentration of fatty acids corresponding to the lipid profile of obese adolescents with metabolic syndrome (OWMS) adolescents was adjusted so as to have the same total amount of FFAs as the serum of normal weight (NW) adolescents (103.25 mg/dL), but the original proportions were conserved (Table 2). MIN6 cells were treated for 24 (A, C, E, G) or 48 hours (B, D, F, H) with the NW or OWMS profiles. Control cells were grown without adding fatty acids. (A, B) The potential of the internal mitochondrial membrane was quantified as the mean intensity of fluorescence (MIF) emitted by MitoTracker during flow cytometry analyses. (C, D) The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was determined using bioluminescence. (E, F) The production of lipid peroxidation products was quantified using the thiobarbituric acid reactive substance assay and is expressed as the concentration of malondialdehyde (MDA) normalized to the total milligram (mg) of protein. (G, H) The capacity to capture peroxynitrite is expressed relative to the antioxidant activity of ascorbic acid (AA) and normalized to the total protein content for each group. AU, arbitrary unit. aIndicates P<0.05, bP<0.01, using analysis of variance followed by Tukey test of multiple comparisons.
Table 1Principal fatty acids in the FFAs of the adolescents in each study group
Fatty acid |
NW |
ONMS |
OWMS |
P valuea
|
Concentration, mg/dL |
No. (%) |
Concentration, mg/dL |
No. (%) |
Concentration, mg/dL |
No. (%) |
Total FFA |
103.25 |
31 (100) |
106.16 |
32 (100) |
121.90 |
32 (100) |
b,c,d
|
C16:0 (palmitic) |
30.01 |
31 (28.71) |
31.85 |
32 (30.00) |
36.75 |
32 (30.15) |
b,c,d
|
C16:1 (palmitoleic) |
1.74 |
8 (1.72) |
1.84 |
19 (1.74) |
2.28 |
27 (1.87) |
NS |
C18:0 (stearic) |
15.60 |
31 (15.16) |
15.30 |
32 (14.41) |
17.30 |
32 (14.20) |
b,c
|
C18:1 n-9c (oleic) |
20.14 |
31 (19.01) |
19.64 |
32 (18.50) |
23.95 |
32 (19.65) |
NS |
C18:2 n-6c (linoleic, LA) |
21.80 |
31 (21.49) |
22.35 |
32 (21.05) |
25.07 |
32 (20.57) |
c,e
|
C20:3 n-6 (cis-8,11,14-dihomo-γ-linolenic, DGLA) |
3.41 |
30 (3.36) |
4.13 |
31 (3.89) |
5.19 |
31 (4.26) |
b,c,d,f
|
C20:4 n (arachidonic, AA) |
6.97 |
31 (7.01) |
7.48 |
31 (7.05) |
8.13 |
32 (6.67) |
b,c
|
C22:6 n-3 (cis-4,7,10,13,16,19-docosahexaenoic, DHA) |
3.59 |
25 (3.54) |
3.56 |
26 (3.36) |
3.21 |
31 (2.63) |
NS |
(LA+DGLA+AA)/DHA |
8.96 |
25 |
9.00 |
26 |
11.96 |
31 |
NS |
Table 2Concentration of FFA in OWMS adolescents, adjusted to the concentration found for NW adolescents
Fatty acid |
NW |
OWMS |
Concentration, mg/dL |
Percentage |
Concentration, mg/dL |
Percentage |
Total FFA |
103.25 |
100.00 |
103.25 |
100.00 |
C16:0 (palmitic) |
30.01 |
28.71 |
31.13 |
30.15 |
C16:1 (palmitoleic) |
1.74 |
1.72 |
1.93 |
1.87 |
C18:0 (stearic) |
15.60 |
15.16 |
14.66 |
14.20 |
C18:1 n-9c (oleic) |
20.14 |
19.01 |
20.29 |
19.65 |
C18:2 n-6c (linoleic, LA) |
21.80 |
21.49 |
21.24 |
20.57 |
C20:3 n-6 (cis-8,11,14-dihomo-γ-linolenic, DGLA) |
3.41 |
3.36 |
4.40 |
4.26 |
C20:4 n (arachidonic, AA) |
6.97 |
7.01 |
6.89 |
6.67 |
C22:6 n-3 (cis-4,7,10,13,16,19-docosahexaenoic, DHA) |
3.59 |
3.54 |
2.72 |
2.63 |
(LA+DGLA+AA)/DHA |
8.96 |
- |
11.96 |
- |