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Moushira Zaki1, Jihan Hussein2, Amr M.M. Ibrahim2 and Eman R. Youness2
1Biological Anthropology Department, Medical Research Division, National Research Centre, Giza, Egypt.
2Medical Biochemistry Department, Medical Research Division, National Research Centre, Giza, Egypt.
Corresponding Author E-mail: jihan_husein@yahoo.com
DOI : https://dx.doi.org/10.13005/bpj/2034
Abstract
Objectives:Elevation of free fatty acids (FFAs) in serum is an importantrisk factor for metabolic changes.Conversely, the relationship between obesity and metabolic abnormalities, and FFAsis not yet completely understood.Thus,we aimed in this study to explore the relationship and the association between insulin resistance (IR), metabolic markers and the variation inplasmaFFAs among the obese women. Methods:This study included fifty obese women aged 25–35 years and has insulin resistance (IR)in addition to fifty age-matched healthy normal weightwomen served as control group.Blood was withdrawn after twelve hours fasting;fasting blood glucose, lipidsand plasma insulinwere estimated;IR was assessedvia the Homeostasis Model Assessment-Insulin Resistance (HOMA-IR).Fatty acids in plasma were measured by HPLC using UV detector that was set at 200 nm.Indeed, anthropometric measurements was performed . Results:Lipid profile, fasting blood sugar, insulin resistance, oleic acids (OA), linoleic acid (LA), arachidonic acid (AA) and anthropometric measurements were significantly increased in IR women compared to control. Whereas, the mean value levels of alpha-linolenicacid(ALA)was significantly decreased in IR women compare to controls. Conclusion:lower plasma levels of ALA and higher levels of AA, OA, LA were significantly associated with risk of IR and metabolic disorder markers in obese women.These results might explain the positive benefits of foods rich with poly unsaturated fatty acids (PUFA).Obesity and IR may be associated with the alterations in composition of the circulating fatty acid.These findings underscore the potential role of PUFA in the metabolic syndrome pathogenesis.
Keywords
Fatty Acids; Insulin Resistance; Metabolic Markers; Obese Women
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| Copy the following to cite this article: Zaki M, Hussein J, Ibrahim A. M. M, Youness E. R. Circulating Plasma Free Fatty Acids, Insulin Resistance and Metabolic Markers in Obese Women. Biomed Pharmacol J 2020;13(4). |
| Copy the following to cite this URL: Zaki M, Hussein J, Ibrahim A. M. M, Youness E. R. Circulating Plasma Free Fatty Acids, Insulin Resistance and Metabolic Markers in Obese Women. Biomed Pharmacol J 2020;13(4). Available from: https://bit.ly/2LXlJub |
Introduction
Obesity is mainly in alliance with the risk of numerous diseases as nonalcoholic fatty liver, cardiovasculardisease (CVD) and diabetes mellitus. When the nutrient intake exceeds the body needs, tissues such as adipose and skeletal and also other body organs like liver become saturated with lipids and resulting in an elevation of lipid export leading to liberation of huge amount of FFAs1 . Previous epidemiologic studies indicated that individuals with higher levels of plasma FFAs were at increased risk for type 2 diabetes (T2D) 2. Free fatty acids (FFAs) are an imperative energy resource human body, and attached to nuclear peroxisomal proliferated-activated receptors (PPARs) interposinggenes expression implicated in the metabolism of both lipidsand glucose3,4. AA ,the omega – 6 fatty acid is found in the cell membrane phospholipids, and the originator of a hugebioactive compounds family called eicosanoids, that are generated via its oxygenation. The liberation of AA from the cell membraneis depending on several enzymes. Additionally, elevation of FFAs levels is linked to insulin resistance through the reduction of glucose transporters and glycogen synthesis5.It was found that plasmaFFA levels are elevated in obese patients and it was hypothesized that increasing of FFAlevels is an important mark of obesity-associated metabolic syndrome. In addition, obesity is associated with elevation of free radicals and oxidative stress that produced as normal endproductsof thecellular metabolism and also during inflammation processby phagocytosis.In adipose tissue insulin resistance leads to increased lipolysis and subsequentlyto increase in the liberation of free FAs, which is the chiefsource of triglycerides stored in the liver.
Consequently, weaimedin this study to give a clear picture about the relationship between insulin resistance and plasma fatty acid in obese women and assess its associations with metabolic markers.
Subjects and Methods
Subjects
This studyisinvolved 100 women(unrelated); 50age-matched healthy women&50 obese women with IR. Theirage was among 21 and 36 years. These cases were indicated from diverse centers to the National Research Centre obesity clinic. The treatise has been authorized by the Ethical Committee of NRC, Egypt (number: 16361), in agreement with the World Medical Association’s Declaration of Helsinki.
Methods
Clinical and biochemical parameters
BMI(Body mass index) was calculated as weight in kilograms divided by height in meters square (kg/m2). MUAC(Mid upper arm circumference) was measured by a resilient tape at the midway between acromial process on the upper right arm with the elbow flexed 90oand the olecranon. Hip circumference (HC)and Waist circumference (WC) were measured in cm. Waist-to-hip ratio (WHR) was calculated. Fat mass was measured by Tanita Body Composition Analyzer (SC-330).
After 12 hours fasting, blood was collected from allpatients, and serum was separated. Blood glucose(fasting)was assessed immediately by enzymatic colorimetric methodCentronic, Germany 6. Insulin level was assessedby ELISA. Whereas, insulin resistance (HOMA-IR) was calculated from the formula: Fasting plasma glucose (mmol/l) period serum insulin level(mU/l) /405. High HOMA-IR values referred tohigh insulin resistance, whereas Low HOMA-IR values indicate high insulinsensitivity as described previously7 .
Aspartate amino transferase(AST)and alanine amino transferase (ALT) in serum were assessed using commercial kit from BioMed Diagnostics according to the method described by8 .
Serum triglycerides (TG) and serum total cholesterol (TC) were determinedby enzymatic colorimetric method. Additionally, high-density lipoprotein cholesterol (HDL-C) wasestimated. Dependently low-density lipoprotein cholesterol (LDL-C) was calculated from the equation mentioned before9 as follow: LDL – C = TC – (HDL- C + TG/5)
Estimation of fatty acids using HPLC
Fractions of fatty acids were assessed sing HPLC, Agilent technologies 1100, equipped with a quaternary pump (model G131A) as described previously10,11.
Fatty acids HPLC standards grade (LA, ALA, OA, AA, DHA) were purchased from Sigma Chemical (Munich, Germany). Acetonitrile, methanol, ethanol, N-hexane, 2-propanol and other laboratory chemicals in this study were HPLC grade. Ultra-pure water was used for all experimental work and analysis12.
Sample preparation
Plasma was homogenized in a solution consists of 2 % acetic acid: ethyl ether mixture (2:1) v/v. This solution was centrifuged at 3000 rpm using cooling centrifuge; the organic layer was evaporated under nitrogen gas untilcomplete dryness. Theresultant residue dissolved in acetonitrile (400 μl)and filtered using hydrophilic PVDF 0.45 μ m before injection.
HPLC condition
Thetechnique was done by RP(reversed phase) HPLC column (260 X 4.6, particle size 5μl) and the used mobile phase was consisted of 70 % acetonitrileby isocratic elution by flow rate 1 ml/min and ;UV detector was at 200 nm. Sequential dilutions of each standard were injected and their corresponding peak zones were specified. The mean values of each fatty acid in all samples were calculated from the linear standard curve.
Statistical Analysis
We performed the statistical analyses using SPSS16.0 for Windows (SPSS Inc). Two-tailed P<0.05 was considered statistically significant.
Results
Table 1 displayed significant differences in anthropometric parameters between IR cases and controls. Obese IR women had significantly higher levels of BMI, body fat %, MUAC and WC than controls (p<.05). In addition, no significant changes were observed in fasting blood sugar, lipid profile, and liver functions between the two studied groups; however insulin and insulin resistance were significantly augmented in obese women compared to control (table 2, 3).
Table 1: Anthropometric measurements in studied groups.
|
Variables
|
Group | Mean ± SD | P value |
| Age | Controls | 33.67±10.735 |
0.121
|
| IR | 36.24 ± 9.595 | ||
| Body mass index (BMI) | Controls | 23.05 ± 4.65 |
0.05
|
| IR | 28.01± 6.63 | ||
| Body fat % | Controls | 23.71 ± 8.61 |
0.001
|
| IR | 35.52 ±12.93 | ||
| Mid upper arm circumference (MUAC) | Controls | 30.66 ± 3.25 |
0.001
|
| IR | 34.04 ± 4.87 | ||
| WC | Controls | 89.17 ± 11.73 |
0.001
|
| IR | 100.93 ± 14.55 | ||
| WHR | Controls | .829 ± 0.07 |
0.33
|
| IR | .840 ± 0.067 |
All data are expressed as mean± SD
P: significant difference (<0.05) in insulin resistance ( IR) group compared to control
P: High significant difference (<0.001) in insulin resistance ( IR) group compared to control
Table 2: Fasting blood sugar, insulin resistance and insulin in studied groups.
| Variables |
Group
|
Mean ± SD | P value |
| FBG (mg/dL) | Controls | 93.45 ± 33.61 |
0.49
|
| IR | 97.84 ± 41.76 | ||
| Insulin( IU/ml) | Controls | 10.3 ±4.9 |
0.05
|
| IR | 16.7 ±5.1 | ||
| HOMA | Controls | 3.3 ± 1.2 |
0.05
|
| IR | 6.4 ± 2.5 |
All data are expressed as mean± SD
P: significant difference (<0.05) in insulin resistance ( IR) group compared to control
P: High significant difference (<0.001) in insulin resistance ( IR) group compared to control
Table 3: Liver functions and lipid profile in studied groups.
| Variables |
Group
|
Mean ± SD | P value |
| ALT (U/L) | Controls | 15.38 ± 8.50 |
0.08
|
| IR | 19.23 ± 18.07 | ||
| AST (U/L) | Controls | 20.22 ± 5.433 |
0.16
|
| IR | 22.45 ± 13.50 | ||
| TC (mg/dL) | Controls | 197.12 ± 37.38 |
0.85
|
| IR | 195.60 ± 38.43 | ||
| TG (mg/dL) | Controls | 98.86 ± 49.29 |
0.73
|
| IR | 101.60 ± 40.88 | ||
| HDL-C (mg/dL) | Controls | 47.84 ± 11.25 |
0.21
|
| IR | 50.45 ± 13.54 | ||
| LDL-C (mg/dL) | Controls | 128.58 ± 43.45 |
0.72
|
| IR | 125.91 ±43.376 |
All data are expressed as mean± SD
P: significant difference (<0.05) in insulin resistance ( IR) group compared to control.
P: High significant difference (<0.001) in insulin resistance ( IR) group compared to control.
Table 4 appeared significant changes in fatty acids fractionation between obese women and control. Thus, the mean value level of OA,LA,and AA was significantly increased along with a significant reduction in ALA in obese group in comparison to control.
Table 4: Plasma fatty acids (μg/ml ) in studied groups.
| Variables |
Group
|
Mean ± SD | P value |
| Oleic acid (OA)
μg/ml |
Controls | 4.53 ± 3.31 |
0.001
|
| IR | 6.56 ± 3.50 | ||
|
Linoleic acid (LA)
|
Controls | 6.13 ± 5.19 |
0.002
|
| IR | 10.34 ± 4.14 | ||
| Archidonic acid (AA) | Controls | 7.12 ± 4.69 |
0.001
|
| IR | 11.30 ± 4.79 | ||
| alpha-linolenic acid (ALA) | Controls | 4.54 ± 0.27 |
0.001
|
| IR | 2.41± 0.38 |
Discussion
Obesity causes numerous metabolic dysregulations including alteration of lipid profile (cholesterol and triglycerides), besides glucose homeostasis including alteration of insulin and its resistance in addition to deteriorationof pro and anti-inflammatorystatus13,14,15. Owing to the hyperlipolytic properties of the visceral adiposity, surplus visceral fat liberates huge quantity of fatty acids; thus, inflow of fatty acids from visceral adipose tissues to the liver through the portal vein is augmented. Furthermore, Nielsen et al. 16elucidatedthat fatty acid liberation from visceral fat into hepatocytes influencedas visceral fat mass augmented. This leads to elevated fatty acid in hepatocytes. Accordingly, stimulating synthesis and secretion of TGin the liverthrough its integration into TG-rich lipoproteinslike very low-density lipoproteins (VLDLs)17 circulating TG is augmenteddue to cumulatingvisceral fat. Furthermore, both the concentrations of the systemic circulating fatty acids and fatty acids in the portal vein levels observedpositive and significant correlations with visceral adipose tissues. In this work, the mean value levels of omega- 3 fatty acids were significantly decreased in obese women compared to control; whereas the mean value levels of omega6 & omega9 were significantly elevated in obese.
The elevation of omega 6 and 9 fatty acids and also the reduction of omega-3 in obese women in this study are linked to the elevation of insulin resistance as appeared in tables2 and 4.
The composition of fatty acids could clarify a phenomenaincludingthe relationship betweeninsulin and its receptors.It was indicated that,thecell membrane fatty acids composition of insulin target tissues, as skeletal muscle &liver, is animportant factor that affects each of insulin production and its vital actions. Consequently, membranes enrich in omega- 3 fatty acids like AL Ahave a tendency to bind more insulin than membrane enrich in omega-6 and 9 fatty acids. Elevation of free fatty acids like unsaturated and omega6 fatty acids results in increase of the fatty acyl-CoA (FAcyl CoA) and diacylglycerol (DAG) concentrations ,resulting in initiation and activation of protein kinase C isoform (PKC-ε) which leads toelevation of insulin receptor substrate-1(IRS-1) serine phosphorylation. Sequentially a reduction of IRS-1 tyrosine phosphorylation &IRS-1 related phosphatidyleinositol 3-kinase (PI3-K) activity causea reduction of insulin –stimulating glucose transport action18 .
Contrarily, ALAimproved insulin sensitivity viarising the responsibility of glucose transporter -4(GLUT-4),that leads to adevelopment of glucose-6- phosphate19.Indeed, Kato et al.,20stated that GLUT-4 inALA treated mice was betterby 250% whencompared to that in control group.
Concomitantly,Hussein et al.,21indicated that flaxseed oil (a plant source of omega-3 fatty acids) has a positiveimpact on reducing insulin resistance in diabetic animalsviascavenging properties of free radicals & increasing antioxidant enzymes. This impact may be due to the up regulation gene expression of antioxidants enzymes and down regulation gene linked with the establishment offree radicals22.
The composition of fatty acid (FA) in serum lipid esters is a mirror to particular extent thedietary composition ofFA during the last 6 to 8 weeks. The serum FA pattern is also dependenton the metabolism of FA and their endogenous synthesis. Also depends on intrauterine &prenatal programming and genetic variation 23.Low levels of linoleic acid (18:2, n-6) &high levels of palmitic acid (16:0) in plasma are characteristic for individualswith metabolic syndrome and insulin resistance24. Arachidonic acid (AA) acts as a powerful negative modulatorof glucose uptake25 and researches have elucidatedelevatedserum levels of arachidonic acid in diabetic subjects in comparison with normal controls26.Thus, the datahave been in agreement with teresearchesthat haveshown a positive relationship between insulin resistanceandAA20,21.
Conclusions
Obesity and IR may be associated with the alterations in composition of the circulating fatty acid.The current study appeared the association of omega6 and 9 fatty acids with insulin resistance and hyperlipidemia.Additionally, these findings underscore the potential role of UFAs in the MS pathogenesis.
Acknowledgments
This work was corroborated by grant from National Research Centre, Egypt.
Conflict of Interest
All authors declared that they have no conflict of interest.
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