Diabetes Metab J.  2017 Aug;41(4):303-315. 10.4093/dmj.2017.41.4.303.

In Vitro Effect of Fatty Acids Identified in the Plasma of Obese Adolescents on the Function of Pancreatic β-Cells

• Velasquez
• Vasquez JS
• Balcazar N

• KMID: 2392471
• DOI: http://doi.org/10.4093/dmj.2017.41.4.303

Abstract

BACKGROUND
The increase in circulating free fatty acid (FFA) levels is a major factor that induces malfunction in pancreatic β-cells. We evaluated the effect of FFAs reconstituted according to the profile of circulating fatty acids found in obese adolescents on the viability and function of the murine insulinoma cell line (mouse insulinoma [MIN6]).
METHODS
From fatty acids obtained commercially, plasma-FFA profiles of three different youth populations were reconstituted: obese with metabolic syndrome; obese without metabolic syndrome; and normal weight without metabolic syndrome. MIN6 cells were treated for 24 or 48 hours with the three FFA profiles, and glucose-stimulated insulin secretion, cell viability, mitochondrial function and antioxidant activity were evaluated.
RESULTS
The high FFA content and high polyunsaturated ω6/ω3 ratio, present in plasma of obese adolescents with metabolic syndrome had a toxic effect on MIN6 cell viability and function, increasing oxidative stress and decreasing glucose-dependent insulin secretion.
CONCLUSION
These results could help to guide nutritional management of obese young individuals, encouraging the increase of ω-3-rich food consumption in order to reduce the likelihood of deterioration of β-cells and the possible development of type 2 diabetes mellitus.

Keyword

Fatty acids, nonesterified; Insulin-secreting cells; Lipotoxicity; Metabolic syndrome; Obesity

MeSH Terms

Adolescent*
Cell Line
Cell Survival
Diabetes Mellitus, Type 2
Fatty Acids*
Fatty Acids, Nonesterified
Humans
In Vitro Techniques*
Insulin
Insulin-Secreting Cells
Insulinoma
Obesity
Oxidative Stress
Plasma*
Fatty Acids
Fatty Acids, Nonesterified
Insulin

Figure

• 

  Fig. 1 Glucose-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. 2 Mitochondrial 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. 3 Effect 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. 4 Effect 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.

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