Print
The supplementary effects of omega-3 fatty acid alone and in a combination with vitamin D3 on serum leptin levels: A randomized clinical trial on men and women with vitamin D deficiency
expand article infoMahmoud S. Abu-Samak‡, Luai Z. Hasoun‡, Abeer Barham‡, Beisan A. Mohammad§, Ibrahim Mosleh|, Ahmad Aljaberi‡, Shady H. Awwad‡
‡ Applied Science Private University, Amman, Jordan
§ Fakeeh College for Medical Sciences, Jeddah, Saudi Arabia
| Jordan University, Amman, Jordan

Corresponding author: Mahmoud S. Abu-Samak ( m_abusamak@asu.edu.jo )

Academic editor: Georgi Momekov
© 2021 Mahmoud S. Abu-Samak, Luai Z. Hasoun, Abeer Barham, Beisan A. Mohammad, Ibrahim Mosleh, Ahmad Aljaberi, Shady H. Awwad.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Abu-Samak MS, Hasoun LZ, Barham A, Mohammad BA, Mosleh I, Aljaberi A, Awwad SH (2021) The supplementary effects of omega-3 fatty acid alone and in a combination with vitamin D3 on serum leptin levels: A randomized clinical trial on men and women with vitamin D deficiency. Pharmacia 68(3): 517-525. https://doi.org/10.3897/pharmacia.68.e64422
Open Access

Abstract

Purpose: This randomized clinical trial (RCT) was designed to assess the effect of VD3, n-3FA, and their combination on serum leptin levels in people with vitamin D deficiency (VDD).

Subjects and methods: One hundred and forty six participants, were randomly assigned into four groups supplemented with the dose of 50,000 IU VD3 taken weekly (D), 300 mg n-3FA taken daily (Om), and their combination (D+Om) or control (C) for eight weeks. Fasting baseline and follow-up (10 weeks; 8 weeks supplementation plus washout period of 2 weeks) of serum 25 hydroxyvitamin D (25OHD), leptin, glucose, triglycerides (TG), parathyroid hormone (PTH), calcium, and phosphorus were assayed. A paired T-test was used to assess the changes in serum leptin levels over of the follow-up period.

Results: Significant increase in follow-up serum leptin (10.62 ± 7.18 to 14.42 ± 8.29 ng/mL, P = 0.002) and TG (154 ± 84.4 to 200.1 ± 79, P = 0.015) levels were observed in n-3-FA supplemented group. Combination therapy (VD3 plus n-3 FA) significantly increased serum 25OHD (13.49 ± 4.64 to 37.09 ± 11.13 ng/mL, P < 0.001), TG levels (114.3 ± 57.3 to 139.1 ± 60.7 mg/mL, P = 0.007) and insignificantly serum leptin (6.74 ± 4.87 to 8.01 ± 6.77 ng/mL, P = 0.269).

Conclusion: Our study referred that notable elevation in leptin and TG levels might be linked to leptin resistance. However, further RCTs are required to clarify possible consequences resulted from the extensive administration of n-3FA supplements and their combinations with high doses of VD3 supplements on humans’ health.

Keywords

leptin, n-3FA, omega, vitamin D3, diabetes

Introduction

Vitamin D deficiency (VDD), obesity, and diabetes mellitus (DM) are worldwide problems. There has been growing interest in the association of type 2 DM (T2DM) with VDD to obesity (Smith and Singleton 2013). Several studies (Abu-Hasheesh et al. 2010; Al-Amer et al. 2015; Abu-Samak et al. 2019) have demonstrated a high prevalence of T2DM and VDD in Mediterranean countries including Jordan. In this context, Kim et al. (2013) found out that VDD was more prevalent in obese people with inverse association with 25-hydroxy vitamin D (25OHD). On the other hand, several randomized clinical trials (RCTs) (Duggan et al. 2014; Mousa et al. 2020) pointed to a vague association between obesity marker (leptin hormone) and VDD. Centrally, leptin overrides satiety as well as peripherally enhances insulin sensitivity (Monzini 2013). Leptin has anti-diabetogenic effects either via improving insulin resistance (IR) and/or stimulating insulin secretion (D’Souza A et al. 2014). It is classified as an adipokine involved in the IR pathogenesis and development of T2DM (Finucane et al. 2009). Some RCTs (Upreti et al. 2018; Niroomand et al. 2019) showed that the treatment of VDD by VD3 may improve the control of diabetes or decrease the risk of disease. Nevertheless, RCTs that examined VD3 supplements on serum leptin levels were contradictory.

According to Dinca et al. (2016), six RCTs, with different therapy protocols, did not show any effect for VD3 supplements on leptin concentration according to meta-analysis data T2DM For example, Duggan et al. (2014) showed that the effect of a small dose of VD3 (2000 IU/day) for 12 months did not change serum leptin. Furthermore, a recent RCT (Mousa et al. 2020) with a higher dose (100,000 IU single bolus followed by 4,000 IU daily) for 16 weeks, showed no differences in leptin levels. Conversely, Hajimohammadi et al. (2017) mentioned a positive association between leptin and 25OHD levels. Elevated serum leptin was also observed after VD3 intervention conducted on 64 patients with the last stage of kidney failure T2DM (Naini AE et al. 2016) . In turn, leptin levels were significantly decreased after a single dose of VD3 (600,000 IU) in 12 subjects with extreme obesity (BMI > 40 kg/m²) T2DM (Mai S et al. 2017). Based on the above, despite the consensus of several observational studies (Kim et al. 2013; Turer et al. 2013) that linked VDD to obesity, RCTs did not confirm the effects of VD3 supplementations on serum leptin levels as a key link to insulin resistance of T2DM.

On the other hand, conflicting data obtained from RCTs showed that there are no (Poreba et al. 2017; Sedlacek et al. 2019) or insignificant reduction (Hariri et al. 2015) in serum leptin after n-3FA intervention. Farimani et al. (2018) showed similar findings in diabetic patients. Generally, the effects of n-3FA on serum leptin in obese people or diabetics are still unclear and need more clarifications according to many systematic reviews. In the same context, a potential synergistic association between VD3 and n-3FA supplementations has taken increase attention in the recent clinical trials (Manson et al. 2015; Al-Shaer et al. 2019; Pittas et al. 2019). Nevertheless, despite the two supplements are widely recommended for several therapeutic aims, studies that highlight the effects of such combination are still not enough. Cadario et al. (2017) administrated the co-supplementation to sustain the remission of T1DM. It reduced the number of wet nights among 7–15-year-old children with nocturnal enuresis according to Rahmani et al. (2018). Razavi et al. (2017) also pointed that VD3 and n-3FA co-supplementation for 6 weeks had beneficial effects in gestational diabetes mellitus.

Conversely, a recent RCT (Pittas et al. 2019) among a total of 2432 persons, at high risk for T2DM, found out that VD3 supplementation at a dose of 4,000 IU per day did not lower the risk of diabetes. Accordingly, this RCT was designed to assess the co-supplementation effects of VD3 50,000 IU once weekly and n-3FA 300 mg per day on serum leptin in men and women with VDD.

Experimental part

Materials and methods

The current randomized clinical trial (RCT) was authorized by the Institutional Review Board (IRB) committee of Applied Science Private University (ASU) (protocol no. DRGS-4-2018-1) and conducted during the winter season, between December 2018 and March 2019. The RCT was performed according to the declaration of Helsinki. The informed consent was filled from each enrolled participant in this clinical trial. Participants were Jordanian men and women from the ASU community in addition to ASU employees’ relatives with an average age at baseline of the trial was 36.18±9.74 years (range 25 to 55). Eligible participants were enrolled based on a confirmed diagnosis of 25OHD levels deficiency (VDD) by internal medicine consultants in Ibn Al-Haytham hospital laboratories. People previously diagnosed with chronic diseases such as kidney problems were excluded due to the association between prolonged administration of VD3 and kidney stone formation (Jackson et al. 2006). Exclusion criteria from the trial also included participants diagnosed with chronic diseases such as osteoporosis, cancer, endocrine disorder and, thalassemia or who have a documented history of allergic reactions to n-3FA supplementations.

Intervention

The baseline and follow-up measurements of the anthropometric and clinical variables were recorded before and after VD3 and n-3FA supplementation. At the end of the interventional protocol which is 8 weeks, the participants entered a washout period of 2 weeks. Then, follow-up measurements were collected for all the participants. A computer-generated randomization was created by an independent statistician. Eligible participants (n = 188) were allocated into four groups as illustrated in the consort chart (Figure 1): Group 1 (C); Participants received no treatment and served as the control group; Group 2 (D); Participants received 50,000 IU of VD3 in a Hi Dee soft gelatin capsule (United Pharmaceuticals Company (Amman, Jordan) once weekly; Group 3 (Om); Participants received soft gelatin capsule of 1,000 mg of wild salmon and fish oil complex (Jamieson Laboratories., Toronto, ON, Canada) once daily. Each capsule contains 300 mg of omega3-FA, equivalent to 300 mg of n-3FA (180 mg as eicosapentaenoic acid and 120 mg as docosahexaenoic acid [DHA]). Group 4 (D+Om); Participants treated with 50,000 IU VD3 once a week and 1000 mg wild salmon and fish oil complex (contains 300 mg of Ω-3FA) once daily. D+Om participants were advised to leave at least a 4 to 6-hour time interval was followed between administering the two supplements. Therapeutic protocols of VD3 and n-3FA supplements were approved in accordance with the Endocrine Society’s Clinical Guidelines for treating VDD in adults (Holick et al. 2011). A Similar protocol of VD3 over 12 months was conducted on humans and did not produce any toxicity (Binkley et al. 2011). DHA doses used in many previous trials were also comparable to the dose used in this trial protocol without side effects as reported in children taking a dose of 600 mg DHA/day (Richardson et al. 2012). Patients’ adherence to the therapeutic protocol was followed via regular mobile phone text messages.

Figure 1. 

CONSORT flow diagram for the study, indicating the number of subjects screened, recruited, and randomly assigned to the different intervention groups.

Anthropometric measurement

This RCT was conducted at Pharmacy school laboratories/ ASU during the winter of 2018 where the timing of blood sampling is important to minimize seasonal fluctuations of total levels of vitamin D in the blood (Wacker M and Holick MF 2013). Anthropometric parameters including height (Ht), body weight (BW), body mass index (BMI), waist (W) circumference, hip (H) circumference, waist/hip ratio (WHR) were measured at baseline and end of the trial.

Clinical parameters assays: After collection, into labeled Eppendorf tubes, serum measurement of clinical parameters was done at the clinical laboratories of Ibn Al-Haytham hospital Amman, Jordan. Vitamin D: Chemiluminescence immunoassay LIAISON 25-hydroxyvitamin D Assay (DiaSorin) was used to measure a total serum concentration of vitamin D (25OHD). The lower limit of assay approximately was (4 ng/mL) with a 100% cross-reactivity with both metabolites of 25OHD, 25OHD2, and 25OHD3 and thus measures total serum 25OHD content. Leptin: Serum levels of leptin were assayed using the enzyme Immunoassay kit (leptin EIA -5302, DRG Diagnostics, Marburg, Germany). Assay Analytical sensitivity was 0.1 ng/mL. Parathyroid Hormone: Serum levels of parathyroid hormone (PTH) were assayed using the enzyme Immunoassay kit (PTH Intact EIA -3645, DRG Diagnostics, Marburg, Germany). The assay analytical sensitivity of the test was 1.57 pg/mL. Calcium and Phosphorus: Calcium and phosphorus (PO4) concentrations in serum were assayed by spectrophotometry method (Clinical Chemistry RAL Analyzers Clima Plus, Spain) using (CALCIUM-ARSENAZO kit (M11570i-15) and Phosphorus Phosphomolybdate/Uv Kit (M11508i-18, BioSystems, Spain). Glucose and Triglycerides: Serum fasting blood glucose (FBG) and triglyceride (TG) levels were assayed on a Roche Cobas C501 analyzer (GLUC3 application, Roche, Mannheim, Germany) using enzymatic colorimetric method. Serum TG levels were measured using TG -BioSystems kits (M11528i-20, BioSystems, Barcelona, Spain). Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) were measured using specific ALT and AST BioSystems kits (M21533i-03, M11531i-23, Barcelona, Spain). Serum creatinine (Cr) and urea levels were measured using Creatinine kit (M12502i-19) and Urea (M11536i-17) kit, BioSystems, Barcelona, Spain) with detection limits = 0.04 mg/dL and 1.3 mg/dL respectively.

Statistical analysis

The parameter changes at the follow-up period (10 weeks) within each group were assessed by paired Student’s t-test. To investigate if there is any significant difference in the mean values for each parameter between the different groups of trial One Way ANOVA test was used. Post hoc comparisons using the Tukey HSD test were conducted to provide specific analysis on which means are significantly different from each other in the four trial groups, differences were considered significant at P < 0.05. To determine potential factors that influenced leptin at the baseline-end of the trial, multiple linear regression was applied using the stepwise method according to the different groups. Kolmogorov–Smirnov test was used to evaluate the normal distribution of study parameters. Data was approximately close to the normal distribution because of the large sample size of the trial. These statistical analyses were performed using SPSS a Statistical Package for the Social Sciences.

Results

The consort diagram (Figure 1) shows that one hundred and forty-six participants completed all stages of the trial out of one hundred seventy eligible participants. Approximately sixty percent of the participants were females (n=88). Due to the poor medication adherence or complying with blood sampling instructions, there were twenty four participants were dropped out of the study. The parental history of DM showed that 18.5% of the participant’s fathers had DM history whereas 24% of the participant’s mothers were diabetic. Furthermore, 49% of the participants practice morning sun exposure.

Baseline participants’ characteristics

The mean age of all participants at baseline of the trial was 36.18 ± 9.74 years (Table 1). BMI mean was 27.57 ± 5.38 kg/m2 which indicated that the population of the study sample was generally overweight. The mean value of 25OHD for all the trial participants was 16.43 ± 7.34 ng/ml which means they were VD deficient. Any volunteer with a vitamin level equals to or higher than 30 ng/mL was not recruited to participate in this RCT. Descriptive analysis of the mean values for clinical parameters is presented in Table 2, all values were within normal ranges.

Table 1.
Download as
CSV
XLSX

Baseline Descriptive Statistics of the Anthropometric Parameters (n = 146).

Anthropometric Parameter Mean ±SD Range
Age (year) 36.18±9.74 25.00-55.00
Weight (kg) 77.47±16.11 42.00-128.00
Height (cm) 166.33±7.93 148.00-187.00
BMI (kg.m-2) 27.57±5.38 14.48-50.08
Waist (cm) 92.01±14.57 46.00-139.00
Hip (cm) 106.77±12.56 43.00-158.00

Note: SD: Standard deviation; BMI: body mass index.
Table 2.
Download as
CSV
XLSX

Baseline Descriptive Statistics of the Clinical Parameters (n = 146).

Clinical parameter Mean ± SD Range Normal ranges
25OHD (ng/mL) 16.43±7.34 3.80-28.2 30-50
FBG (mg/dL) 84.88±17.79 67.10-115.20 70-110
Leptin (ng/mL) 8.78±6.44 1.10-31.70 *NA
TG (mg/dL) 129.61±67.12 26.30-401.50 Up to 150
PTH (pg/mL) 33.56±10.95 9.26-50.00 9-90
Calcium (mg/dL) 9.86±1.78 8.50-18.60 8.6-10.3
PO4 (mg/dL) 4.12±0.27 3.30-5.10 2.5-4.5
Cr (mg/dL) 0.90±0.07 0.50-1.20 0.5-1.1
ALT (U/L) 9.36±3.43 0.90-33.00 Up to 65
AST (U/L) 7.29±1.72 3.00-18.00 Up to 30
Urea (mmol/L) 3.03±0.73 0.90-7.50 2.5 to 7.1

Note: 25OHD: 25-hydroxy vitamin D; FBG: fasting blood glucose; TG: triglycerides; PTH: parathyroid hormone; PO4: phosphate; Cr: creatinine; ALT: alanine aminotransferase; AST: aspartate aminotransferase. * NA, not applicable.

Correlation of selected obesity variables with 25OHD levels

A significant negative correlation between 25OHD and leptin levels was observed in people with VDD at the baseline of the trial (R = –0.257, P = 0.002). A negative correlation but insignificant was also observed between 25OHD levels and BMI values as shown in Table 3 (R = –0.153, P = 0.066). No significant correlations for the mentioned variables with 25OHD were observed at end of the trial.

Table 3.
Download as
CSV
XLSX

Correlation of selected obesity variables with 25OHD levels at baseline and follow-up.

Obesity variable Baseline Follow up
R P-value R P-value
BMI (kg.m-2) -0.153 0.066 *-0.032 0.705
Weight (kg) -0.003 0.975 **0.112 0.179
Waist (cm) -0.062 0.458 0.072 0.391
Leptin (ng/mL) -0.257 0.002 -0.078 0.347
WHR 0.119 0.154 -0.088 0.291

Note: *: negative correlation, **: positive correlation BMI: body mass index; WHR: waist/hip ratio; R: correlation coefficient.

Follow-up results

25OHD levels

At the end of the trial, significant differences were observed between the baseline and follow-up mean serum 25OHD levels in the three intervention groups (P-value < 0.01) as shown in Table 4. The mean levels of serum 25OHD were significantly increased (PA < 0.01) in D (38.4 ± 13.4 vs 17.42 ± 5.7ng/mL) as well as in D+Om study groups (39.7 ± 11.8 vs 16.95 ± 4.8 ng/mL) . On the other hand, a significant decrease in 25OHD levels was observed in Om study group ((12.34 ± 5.9 vs 19.7 ± 6.03 ng/ml, PA < 0.01).

Table 4.
Download as
CSV
XLSX

Fasting serum 25OHD at baseline and follow-up.

Fasting serum 25OHD (ng/mL)
Group C D Om D+Om P-value
Baseline 18.34 ± 7.2 17.42 ± 5.7 19.7 ± 6.03 16.95 ± 4.8 PB = 0.1
Follow-up 17.7 ± 6.5 38.4 ± 13.4 12.34 ± 5.9 39.7 ± 11.8 PC < 0.001
Change 0.65 -20.95 7.38 -22.75
PA 0.26 < 0.01 < 0.01 < 0.01

Note: Change = baseline mean – follow-up mean PA: P-value for paired t-test between baseline and the follow-up of the study for each group; PB: P-value for ANOVA with post hoc Tukey HSD done at baseline to compare among all groups; PC: P-value for ANOVA with post hoc Tukey HSD (honestly significant difference) done at the follow-up of the study to compare among all groups. Abbreviations: 25OHD: 25-hydroxy vitamin D; C: control group; D: vitamin D3 supplementation group; Om: n-3FA supplementation group; D+Om: vitamin D3 plus n-3FA supplementation group.

Serum leptin levels

Exceptionally, only the Om group (Table 5) showed a significant increase (P = 0.002) in the mean serum leptin levels at the end of the trial compared to baseline (10.62 ± 7.18 ng/mL vs 14.42 ± 8.29 ng/mL). Post-hoc test at follow-up (Suppl. material 1: Table S1) also indicated that the mean levels of leptin in the Om group were significantly increased in comparison with the control group.

Table 5.
Download as
CSV
XLSX

Serum levels of leptin at baseline and follow-up.

Serum leptin ng/ml
Group C D Om D+Om P- value
Baseline 10.11±8.5 8.54±5.1 10.62±7.2 7.74±4.9 P B= 0.083
Follow-up 11.49 ±10.3 10.26±7.6 14.42±8.3 8.01±6.8 P C= 0.005
Change -1.38 -1.72 -3.81 -0.27
P A 0.501 0.083 0.002 0.269

Note: Change = baseline mean – follow-up mean C: control group; D: vitamin D3 supplementation group; Om: n-3FA supplementation group; D+Om: vitamin D3 plus n-3FA supplementation group. PA: P-value for paired t-test between baseline and the follow-up of the study for each group; PB: P-value for ANOVA with post hoc Tukey HSD (honestly significant difference) done at baseline to compare among all groups; PC: P-value for ANOVA with post hoc Tukey HSD done at the follow-up of the study to compare among all groups.

Serum PTH, calcium, and phosphate levels

In the current RCT, compared to baseline levels, no significant changes were noted in serum PTH and calcium at follow-up. In turn, except for the C group, phosphorus levels showed notable changes in all study groups with a significant decrease in the means of serum phosphorus (Suppl. material 1: Table S2).

Serum TG levels

The mean serum TG levels were significantly increased in both Om and D+Om groups (P-value = 0.015, 0.007, respectively) as shown in Table 6.

Table 6.
Download as
CSV
XLSX

Serum TG levels at Baseline and at End of the Trial.

Group Control D Om D+Om p-Value
TG Baseline 120.1±48.6 130.9±65.5 154.0±84.4 114.3±57.3 P B= 0.058
Follow-up 113.4±53.8 137.3±85.3 200.1±79 139.1±60.7 P C < 0.05
Change 6.7 -6.4 -46.1 -24.8
PA 0.328 0.608 0.015 0.007

Note: Change = baseline mean – follow-up mean C: control group; D: vitamin D3 supplementation group; Om: n-3FA supplementation group; D+Om: vitamin D3 plus n-3FA supplementation group; TG: Triglycerides. PA: P-value for paired t-test between baseline and the follow-up of the study for each group; PB: P-value for ANOVA with post hoc Tukey HSD (honestly significant difference) done at baseline to compare among all groups; PC: P-value for ANOVA with post hoc Tukey HSD done at the follow-up of the study to compare among all groups.

Stepwise regression analysis

The multivariate stepwise regression analysis showed that the predictors at the period of follow-up, that are included in the model together explained ~50%, 33%, 13%, and 49% of the variance in leptin levels in the C, D, Om, and D+Om study groups, respectively (Table 7).

Table 7.
Download as
CSV
XLSX

The Multivariate Association between the Trial Variables and Serum Levels of Leptin Using Stepwise Regression at the period of the follow-up.

Dependent variable Study group Univariate effect estimates Coefficient
B F R R2
Leptin (ng/mL) C Calcium (mg/dL) **3.157 14.245 0.610 0.372
ALT (U/L) 1.741 11.510 0.707 0.500
D Paternal DM *-5.433 10.712 0.484 0.234
Weight (kg) -0.125 8.312 0.573 0.328
Om AST (U/L) 1.677 5.088 0.356 0.127
D+Om Calcium (mg/dL) 1.434 12.476 0.470 0.221
PTH (pg/mL) 0.123 10.295 0.569 0.324
Height (cm) -0.199 8.989 0.625 0.391
Gender -3.841 8.55 0.674 0.455
Sun-exposure 0.229 15.336 0.685 0.469
HtWt R (cm/Kg) 0.005 13.510 0.701 0.491

Note: *: negative correlation, **: positive correlation C: control group; D: vitamin D3 supplementation group; Om: n-3FA supplementation group; D+Om: vitamin D3 plus n-3FA supplementation group; ALT: alanine aminotransferase; DM: Diabetes Mellitus.

Discussion

This novel clinical trial on people with VDD indicated that 300 mg daily for 8 weeks of n-3FA significantly raised serum leptin levels concurrently with the decrease of the 25OHD levels. Our observations are in agreement with previous RCTs (Gray et al. 2013; Huang et al. 2019) showed a positive association between leptin and n-3FA levels in obese participants. Consistent with the results of a previous study (Forsythe et al. 2012), baseline findings of the current RCT indicated an inverse association between VDD and serum leptin. It was proposed that adipose tissue absorbs the fat-soluble VD (Wortsman et al. 2000) which may explain an inverse correlation also seen by Vimaleswaran et al (2013) between fat mass and serum 25OHD levels. Long-term regulation of body weight is mainly affected by the degree of obesity (Latner et al. 2000; Montesi et al. 2016). Leptin secretion from fat cells is proportional to the number of stored triglycerides (Park et al. 2016). In the Om group, a significant increase of both TG and leptin levels, at the end of our trial, supports Park et al. (2016) assumption. Later, Vanavanan et al. (2017) correlated between long-term supplementation of n-3FA and the excess ingestion of calories which may cause an increase in fat deposition and prompts an increase in leptin secretion. In this context, overfeeding is known as an inducer of leptin secretion which is strongly associated with insulin resistance (Wang et al. 2001). Insulin resistance in the term of central adiposity is a key link between obesity with T2DM (Gonzalez 2012). This is believed to be essential for the pathogenesis of metabolic syndrome in pre-diabetics, which is characterized by hyperinsulinemia and hypertriglyceridemia (Hirano et al. 2018). Insulin resistance progression could be associated with dysregulated secretion of the anti-diabetogenic activity of leptin (D’Souza et al. 2014). Therefore leptin hypersecretion is a sign of the presence of leptin resistance in the peripheral tissues that develop as obesity progress and early development of IR (Kloting et al. 2014). Insulin insensitivity induced by hyperleptinemia may increase the activity of hormone-sensitive lipase, the enzyme that hydrolyzes stored TG. Concerning the combination of n-3FA and VD3, a significant elevation of TG levels in the D+Om group was accompanied by an insignificant elevation of leptin levels. We propose that the existence of a synergistic effect for the combination of VD3 and n-3FA leads to the elevation of serum TG in people with VDD. Elevated 25OHD levels seen in the D+Om group could be involved in dysregulated leptin secretion. However, it is not clear whether central or peripheral action of the supplement was involved in that mentioned association.

Insulin resistance often associates with exhausted pancreatic beta cells due to prolonged insulin hypersecretion because of poor glycemic control in diabetics or prolonged hyperglycemic levels in pre-diabetics (Al-Shoumer et al. 2015). Insulin hypersecretion subsequently down-regulates insulin receptors causing the development of IR in obese subjects (Xu et al. 2007). Likewise, the co-supplementation of VD3 plus n-3FA perhaps mimics a potential mechanism that stimulates insulin synthesis via VDR in beta cells (Baidal et al. 2016) provoking insulin receptor downregulation. In mice fed with a diet enriched with eicosapentaenoic acid (EPA), higher insulin secretion did not improve glucose tolerance in induced T2DM animal models (Neuman et al. 2017). Unfortunately, due to logistic reasons in this RCT, we did not measure the specific glycemic control parameters including insulin, c-peptide or glycated hemoglobin (HbA1c), and these can be considered a trial limitation. Furthermore, one of the limitation that we faced in this study is that, during the trial, there was a significant drop in the participants of control group which was explained previously in the consort diagram. Other studies were inconsistent either due to dose-dependent (Gray et al. 2013; Poreba et al. 2017), duration of therapy protocol (Jacobo-Cejudo et al. 2017), or the sample allocated for the clinical trial (Hariri et al. 2015). For instance, Poreba et al. (2017) showed that n-3FA therapy with a 520 mg dose for 3 months or with 2gm for 6 weeks did not change leptin levels in T2DM patients. In the systematic review (Farimani et al. 2018) of 10 RCTs involved 494 participants with T2DM treated with doses of (520–740 mg/day n-3FA) an insignificant reduction in leptin levels were observed. However, Farimani et al. (2018) concluded that these RCTs had not reached a definitive conclusion about the effect of n-3FA on the leptin levels in T2DM. Finally, the same dose, 520 mg of DHA + EPA, but for 24 weeks on 54 T2DM patients showed a significant reduction in leptin levels (Jacobo-Cejudo et al. 2017). Remarkably, a significant increase in insulin levels observed by Jacobo-Cejudo et al (2017) has been linked anti- diabetogenic effect of n-3FA supplementation in T2DM patients. However, elevated insulin levels are accompanied by prolonged hyperglycemia (Heianza et al. 2012). Overall, it is unclear whether n-3FA or its combination with VD3 has anti-diabetogenic effects in people with VDD.

Conclusion

This novel RCT provides that eight weeks of n-3FA therapy significantly declined 25OHD levels which were accompanied by a significant increase in leptin and TG levels. Although the combined effect of VD3 plus n-3FA, at follow-up, normalized 25OHD levels, notable elevation in TG, and leptin levels were seen. Accordingly, the intervention of VD3, n-3FA, or their combination may be accompanied by negative effects on leptin resistance. Further clinical trials are required to clarify possible consequences resulted from the co-supplementation of VD3 and omega on humans’ health.

Acknowledgments

The authors are grateful to the Applied Science Private University (ASU), Amman, Jordan, for the full financial support granted for this research.

References

  • Abu-Samak MS, AbuRuz ME, Masa’Deh R, Khuzai R, Jarrah S (2019) Correlation of selected stress associated factors with vitamin D deficiency in Jordanian men and women. International Journal of General Medicine 12: 225–233. https://doi.org/10.2147/IJGM.S198175 [PMID: 31303782; PMCID: PMC6612048]
  • Abu-Hasheesh MO, Abu-Samak MS, Al-Matubsi HY, Jaradeh MS, Jarrah SS, Khuzaie RF (2010) Association of parental history of type 2 diabetes mellitus with leptin levels in Jordanian male youths. Saudi Medical Journal 31(8): 882–886. [PMID: 20714685]
  • Acobo-Cejudo MG, Valdés-Ramos R, Guadarrama-López AL, Pardo-Morales RV, Martínez-Carrillo BE, Harbige LS (2017) Effect of n-3 Polyunsaturated Fatty Acid Supplementation on Metabolic and Inflammatory Biomarkers in Type 2 Diabetes Mellitus Patients. Nutrients 9(6): e573. https://doi.org/10.3390/nu9060573 [PMID: 28587203; PMCID: PMC5490552]
  • Al-Shaer AH, Abu-Samak MS, Hasoun LZ, Mohammad BA, Basheti IA (2019) Assessing the effect of omega-3 fatty acid combined with vitamin D3 versus vitamin D3 alone on estradiol levels: a randomized, placebo-controlled trial in females with vitamin D deficiency. Clinical Pharmacology 11: 25–37. https://doi.org/10.2147/CPAA.S182927 [PMID: 30787641; PMCID: PMC6366354]
  • Baidal DA, Ricordi C, Garcia-Contreras M, Sonnino A, Fabbri A (2016) Combination high-dose omega-3 fatty acids and high-dose cholecalciferol in new onset type 1 diabetes: a potential role in preservation of beta-cell mass. European Review for Medical and Pharmacological Sciences 20(15): 3313–3318. [PMID: 27467009]
  • Binkley N, Gemar D, Engelke J, Gangnon R, Ramamurthy R, Krueger D, Drezner MK (2011) Evaluation of ergocalciferol or cholecalciferol dosing, 1,600 IU daily or 50,000 IU monthly in older adults. The Journal of Clinical Endocrinology and Metabolism 96(4): 981–988. https://doi.org/10.1210/jc.2010-0015 [Epub 2011 Feb 2. PMID: 21289249; PMCID: PMC3417158]
  • Cadario F, Savastio S, Rizzo AM, Carrera D, Bona G, Ricordi C (2017) Can Type 1 diabetes progression be halted? Possible role of high dose vitamin D and omega 3 fatty acids. European Review for Medical and Pharmacological Sciences 21(7): 1604–1609. [PMID: 28429367]
  • Dinca M, Serban MC, Sahebkar A, Mikhailidis DP, Toth PP, Martin SS, Blaha MJ, Blüher M, Gurban C, Penson P, Michos ED, Hernandez AV, Jones SR, Banach M (2016) For Lipid Blood Pressure Meta-Analysis Collaboration LBPMC Group. Does vitamin D supplementation alter plasma adipokines concentrations? A systematic review and meta-analysis of randomized controlled trials. Pharmacological Research 107: 360–371. https://doi.org/10.1016/j.phrs.2016.03.035 [Epub 2016 Mar 30. PMID: 27038530]
  • D’souza AM, Asadi A, Johnson JD, Covey SD, Kieffer TJ (2014) Leptin deficiency in rats results in hyperinsulinemia and impaired glucose homeostasis. Endocrinology 155(4): 1268–1279. https://doi.org/10.1210/en.2013-1523 [Epub 2014 Jan 27. PMID: 24467741]
  • Duggan C, de Dieu Tapsoba J, Mason C, Imayama I, Korde L, Wang CY, McTiernan A (2015) Effect of Vitamin D3 Supplementation in Combination with Weight Loss on Inflammatory Biomarkers in Postmenopausal Women: A Randomized Controlled Trial. Cancer Prevention Research 8(7): 628–635. https://doi.org/10.1158/1940-6207.CAPR-14-0449 [CAPR-14-0449. Epub 2015 Apr 23. PMID: 25908506; PMCID: PMC4491001]
  • Farimani AR, Hariri M, Azimi-Nezhad M, Borji A, Zarei S, Hooshmand E (2012) The effect of n-3 PUFAs on circulating adiponectin and leptin in patients with type 2 diabetes mellitus: a systematic review and meta-analysis of randomized controlled trials. Acta Diabetologica 55(7): 641–652. https://doi.org/10.1007/s00592-018-1110-6 [Epub 2018 Feb 16. PMID: 29453672]
  • Finucane FM, Luan J, Wareham NJ, Sharp SJ, O’Rahilly S, Balkau B, Flyvbjerg A, Walker M, Højlund K, Nolan JJ, Savage DB (2009) Correlation of the leptin: adiponectin ratio with measures of insulin resistance in non-diabetic individuals. Diabetologia 52(11): 2345–2349. https://doi.org/10.1007/s00125-009-1508-3 [Epub 2009 Sep 12. PMID: 19756488; PMCID: PMC2759015]
  • Forsythe LK, Livingstone MB, Barnes MS, Horigan G, McSorley EM, Bonham MP, Magee PJ, Hill TR, Lucey AJ, Cashman KD, Kiely M, Strain JJ, Wallace JM (2012) Effect of adiposity on vitamin D status and the 25-hydroxycholecalciferol response to supplementation in healthy young and older Irish adults. British Journal of Nutrition 107(1): 126–134. https://doi.org/10.1017/S0007114511002662 [Epub 2011 Jun 28. PMID: 21733320]
  • Gray B, Steyn F, Davies PS, Vitetta L (2013) Omega-3 fatty acids: a review of the effects on adiponectin and leptin and potential implications for obesity management. European Journal of Clinical Nutrition 67(12): 1234–1242. https://doi.org/10.1038/ejcn.2013.197 [Epub 2013 Oct 16. PMID: 24129365]
  • Hajimohammadi M, Shab-Bidar S, Neyestani TR (2017) Vitamin D and serum leptin: a systematic review and meta-analysis of observational studies and randomized controlled trials. European Journal of Clinical Nutrition 71(10): 1144–1153. https://doi.org/10.1038/ejcn.2016.245 [Epub 2016 Dec 14. PMID: 27966571]
  • Hariri M, Ghiasvand R, Shiranian A, Askari G, Iraj B, Salehi-Abargouei A (2015) Does omega-3 fatty acids supplementation affect circulating leptin levels? A systematic review and meta-analysis on randomized controlled clinical trials. Clin Endocrinol 82(2): 221–228. https://doi.org/10.1111/cen.12508 [Epub 2014 Jul 7. PMID: 24862919]
  • Heianza Y, Arase Y, Fujihara K, Tsuji H, Saito K, Hsieh SD, Kodama S, Shimano H, Yamada N, Hara S, Sone H (2012) High normal HbA(1c) levels were associated with impaired insulin secretion without escalating insulin resistance in Japanese individuals: the Toranomon Hospital Health Management Center Study 8 (TOPICS 8). Diabetic Medicine 29(10): 1285–1290. https://doi.org/10.1111/j.1464-5491.2012.03667.x [PMID: 22486679]
  • Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH, Weaver CM (2011) Endocrine Society. Evaluation, treatment, and prevention of vitamin D deficiency: An Endocrine Society clinical practice guideline. The Journal of Clinical Endocrinology and Metabolism 96(7): 1911–1930. https://doi.org/10.1210/jc.2011-0385 [Epub 2011 Jun 6. Erratum in: J Clin Endocrinol Metab. 2011 Dec;96(12):3908. PMID: 21646368]
  • Huang F, Del-Río-Navarro BE, Leija-Martinez J, Torres-Alcantara S, Ruiz-Bedolla E, Hernández-Cadena L, Barraza-Villarreal A, Romero-Nava R, Sanchéz-Muñoz F, Villafaña S, Marchat LA, Hong E (2019) Effect of omega-3 fatty acids supplementation combined with lifestyle intervention on adipokines and biomarkers of endothelial dysfunction in obese adolescents with hypertriglyceridemia. Journal of Nutritional Biochemistry 64: 162–169. https://doi.org/10.1016/j.jnutbio.2018.10.012 [Epub 2018 Nov 7. PMID: 30513433]
  • Jackson RD, LaCroix AZ, Gass M, Wallace RB, Robbins J, Lewis CE, Bassford T, Beresford SA, Black HR, Blanchette P, Bonds DE, Brunner RL, Brzyski RG, Caan B, Cauley JA, Chlebowski RT, Cummings SR, Granek I, Hays J, Heiss G, Hendrix SL, Howard BV, Hsia J, Hubbell FA, Johnson KC, Judd H, Kotchen JM, Kuller LH, Langer RD, Lasser NL, Limacher MC, Ludlam S, Manson JE, Margolis KL, McGowan J, Ockene JK, O’Sullivan MJ, Phillips L, Prentice RL, Sarto GE, Stefanick ML, Van Horn L, Wactawski-Wende J, Whitlock E, Anderson GL, Assaf AR, Barad D (2006) Calcium plus vitamin D supplementation and the risk of fractures. The New England Journal of Medicine 354(7): 669–683. https://doi.org/10.1056/NEJMoa055218 [Erratum in: N Engl J Med. 2006 Mar 9;354(10):1102. PMID: 16481635]
  • Kim M, Na W, Sohn C (2013) Correlation between vitamin D and cardiovascular disease predictors in overweight and obese Koreans. Journal of Clinical Biochemistry and Nutrition 52(2): 167–171. https://doi.org/10.3164/jcbn.12-81 [Epub 2013 Feb 22. PMID: 23524629; PMCID: PMC3593135]
  • Lanzerstorfer P, Yoneyama Y, Hakuno F, Müller U, Höglinger O, Takahashi S, Weghuber J (2015) Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces. The FEBS Journal 282(6): 987–1005. https://doi.org/10.1111/febs.13213 [Epub 2015 Feb 11. PMID: 25627174]
  • Latner JD, Stunkard AJ, Wilson GT, Jackson ML, Zelitch DS, Labouvie E. (2000) Effective long-term treatment of obesity: a continuing care model. International Journal Of Obesity And Related Metabolic Disorders 24(7): 893–898. https://doi.org/10.1038/sj.ijo.0801249 [PMID: 10918537]
  • Mai S, Walker GE, Vietti R, Cattaldo S, Mele C, Priano L, Mauro A, Bona G, Aimaretti G, Scacchi M, Marzullo P (2017) Acute Vitamin D₃ Supplementation in Severe Obesity: Evaluation of Multimeric Adiponectin. https://doi.org/10.3390/nu9050459
  • Manson JE, Bassuk SS, Lee IM, Cook NR, Albert MA, Gordon D, Zaharris E, Macfadyen JG, Danielson E, Lin J, Zhang SM, Buring JE (2012) The VITamin D and OmegA-3 TriaL (VITAL): rationale and design of a large randomized controlled trial of vitamin D and marine omega-3 fatty acid supplements for the primary prevention of cancer and cardiovascular disease. Contemporary Clinical Trials 33(1): 159–171. https://doi.org/10.1016/j.cct.2011.09.009 [Epub 2011 Oct 2. PMID: 21986389; PMCID: PMC3253961]
  • Montesi L, El Ghoch M, Brodosi L, Calugi S, Marchesini G, Dalle Grave R (2016) Long-term weight loss maintenance for obesity: a multidisciplinary approach. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 9: 37–46. https://doi.org/10.2147/DMSO.S89836 [PMID: 27013897; PMCID: PMC4777230]
  • Mousa A, Naderpoor N, Wilson K, Plebanski M, de Courten MPJ, Scragg R, de Courten B (2020) Vitamin D supplementation increases adipokine concentrations in overweight or obese adults. European Journal of Nutrition 59(1): 195–204. https://doi.org/10.1007/s00394-019-01899-5 [Epub 2019 Jan 16. PMID: 30649593]
  • Naini AE, Vahdat S, Hedaiati ZP, Shahzeidi S, Pezeshki AH, Nasri H (2016) The effect of vitamin D administration on serum leptin and adiponectin levels in end-stage renal disease patients on hemodialysis with vitamin D deficiency: A placebo-controlled double-blind clinical trial. Journal of Research in Medical Sciences 21: 1–1. https://doi.org/10.4103/1735-1995.175144 [PMID: 27904547; PMCID: PMC5122181]
  • Neuman JC, Schaid MD, Brill AL, Fenske RJ, Kibbe CR, Fontaine DA, Sdao SM, Brar HK, Connors KM, Wienkes HN, Eliceiri KW, Merrins MJ, Davis DB, Kimple ME (2017) Enriching Islet Phospholipids with Eicosapentaenoic Acid Reduces Prostaglandin E2 Signaling and Enhances Diabetic β-Cell Function. Diabetes 66(6): 1572–1585. https://doi.org/10.2337/db16-1362 [Epub 2017 Feb 13. PMID: 28193789; PMCID: PMC5440023]
  • Niroomand M, Fotouhi A, Irannejad N, Hosseinpanah F (2019) Does high-dose vitamin D supplementation impact insulin resistance and risk of development of diabetes in patients with pre-diabetes? A double-blind randomized clinical trial. Diabetes Research and Clinical Practice 148: 1–9. https://doi.org/10.1016/j.diabres.2018.12.008 [Epub 2018 Dec 21. PMID: 30583032]
  • Pittas AG, Dawson-Hughes B, Sheehan P, Ware JH, Knowler WC, Aroda VR, Brodsky I, Ceglia L, Chadha C, Chatterjee R, Desouza C, Dolor R, Foreyt J, Fuss P, Ghazi A, Hsia DS, Johnson KC, Kashyap SR, Kim S, LeBlanc ES, Lewis MR, Liao E, Neff LM, Nelson J, O’Neil P, Park J, Peters A, Phillips LS, Pratley R, Raskin P, Rasouli N, Robbins D, Rosen C, Vickery EM, Staten M (2019) Vitamin D Supplementation and Prevention of Type 2 Diabetes. The New England Journal of Medicine 381(6): 520–530. https://doi.org/10.1056/NEJMoa1900906 [Epub 2019 Jun 7. PMID: 31173679; PMCID: PMC6993875]
  • Poreba M, Mostowik M, Siniarski A, Golebiowska-Wiatrak R, Malinowski KP, Haberka M, Konduracka E, Nessler J, Undas A, Gajos G (2017) Treatment with high-dose n-3 PUFAs has no effect on platelet function, coagulation, metabolic status, or inflammation in patients with atherosclerosis and type 2 diabetes. Cardiovascular Diabetology 16(1): 1–50. https://doi.org/10.1186/s12933-017-0523-9 [PMID: 28410617; PMCID: PMC5391604]
  • Rahmani E, Eftekhari MH, Fallahzadeh MH, Fararouei M, Massoumi SJ (2018) Effect of vitamin D and omega-3 on nocturnal enuresis of 7–15-year-old children. Journal of Pediatric Urology 14(3): 257.e1–257.e6. https://doi.org/10.1016/j.jpurol.2018.01.007 [Epub 2018 Feb 5. PMID: 29598883]
  • Razavi M, Jamilian M, Samimi M, Afshar Ebrahimi F, Taghizadeh M, Bekhradi R, Seyed Hosseini E, Haddad Kashani H, Karamali M, Asemi Z (2017) The effects of vitamin D and omega-3 fatty acids co-supplementation on biomarkers of inflammation, oxidative stress and pregnancy outcomes in patients with gestational diabetes. Nutrition & Metabolism 14: 1–80. https://doi.org/10.1186/s12986-017-0236-9 [PMID: 29299042; PMCID: PMC5745759]
  • Richardson AJ, Burton JR, Sewell RP, Spreckelsen TF (2012) Docosahexaenoic acid for reading, cognition and behavior in children aged 7–9 years: a randomized, controlled trial (the DOLAB Study). PLoS ONE 7(9): e43909. https://doi.org/10.1371/journal.pone.0043909 [Epub 2012 Sep 6. PMID: 22970149; PMCID: PMC3435388]
  • Sedláček P, Plavinová I, Langmajerová J, Dvořáková J, Novák J, Trefil L, Müller L, Buňatová P, Zeman V, Müllerová D (2018) Effect of n-3 fatty acids supplementation during lifestyle modification in women with overweight. Central European Journal of Public Health 26(4): 265–271. https://doi.org/10.21101/cejph [PMID: 30660136]
  • Upreti V, Maitri V, Dhull P, Handa A, Prakash MS, Behl A (2018) Effect of oral vitamin D supplementation on glycemic control in patients with type 2 diabetes mellitus with coexisting hypovitaminosis D: A parellel group placebo controlled randomized controlled pilot study. Diabetology & Metabolic Syndrome 12(4): 509–512. https://doi.org/10.1016/j.dsx.2018.03.008 [Epub 2018 Mar 16. PMID: 29580871.24. PMID: 23266927]
  • Vanavanan S, Srisawasdi P, Rochanawutanon M, Kumproa N, Kruthkul K, Kroll MH (2018) Performance of body mass index and percentage of body fat in predicting cardiometabolic risk factors in Thai adults. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 11: 241–253. https://doi.org/10.2147/DMSO.S167294 [PMID: 29910627; PMCID: PMC5987789]
  • Vimaleswaran KS, Berry DJ, Lu C, Tikkanen E, Pilz S, Hiraki LT, Cooper JD, Dastani Z, Li R, Houston DK, Wood AR, Michaëlsson K, Vandenput L, Zgaga L, Yerges-Armstrong LM, McCarthy MI, Dupuis J, Kaakinen M, Kleber ME, Jameson K, Arden N, Raitakari O, Viikari J, Lohman KK, Ferrucci L, Melhus H, Ingelsson E, Byberg L, Lind L, Lorentzon M, Salomaa V, Campbell H, Dunlop M, Mitchell BD, Herzig KH, Pouta A, Hartikainen AL, Streeten EA, Theodoratou E, Jula A, Wareham NJ, Ohlsson C, Frayling TM, Kritchevsky SB, Spector TD, Richards JB, Lehtimäki T, Ouwehand WH, Kraft P, Cooper C, März W, Power C, Loos RJ, Wang TJ, Järvelin MR, Whittaker JC, Hingorani AD, Hyppönen E (2013) Causal relationship between obesity and vitamin D status: bi-directional Mendelian randomization analysis of multiple cohorts. PLoS Medicine 10(2): e1001383. https://doi.org/10.1371/journal.pmed.1001383 [Epub 2013 Feb 5. PMID: 23393431; PMCID: PMC3564800]
  • Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF (2003) Decreased bioavailability of vitamin D in obesity. The American Journal of Clinical Nutrition 72(3): 690–693. https://doi.org/10.1093/ajcn/72.3.690 [Erratum in: Am J Clin Nutr. 77(5):1342. PMID: 10966885]
  • Xu G, Kaneto H, Laybutt DR, Duvivier-Kali VF, Trivedi N, Suzuma K, King GL, Weir GC, Bonner-Weir S (2007) Downregulation of GLP-1 and GIP receptor expression by hyperglycemia: possible contribution to impaired incretin effects in diabetes. Diabetes 56(6): 1551–1558. https://doi.org/10.2337/db06-1033 [Epub 2007 Mar 14. PMID: 17360984]
login to comment