Pomegranate Juice Increases Sirtuin1 Protein in Peripheral Blood Mononuclear Cell from Patients with Type 2 Diabetes: A Randomized Placebo Controlled Clinical Trial

Abstract

Background:

Few studies have examined anti-inflammatory effects of pomegranate juice (PJ). The present study aimed to evaluate the effect of PJ on nuclear factor kappa B (NF-κB) p65 and sirtuin1 in peripheral blood mononuclear cell (PBMC), and plasma vascular inflammation biomarkers.

Methods:

Patients with type 2 diabetes were randomly assigned to either the PJ (n = 22) or the placebo group (n = 22). The patients in the PJ group received 250 mL of PJ daily for 12 weeks, whereas the placebo group received corresponding control beverages of similar color and energy content. At baseline and at the end of week 12, fasting plasma concentrations of soluble intercellular adhesion molecule type 1 (sICAM-1), soluble vascular cell adhesion molecule type 1 (sVCAM-1), and soluble E-selectin (sE-selectin) were measured. NF-κB p65 and SIRT1 were measured in the PBMC.

Results:

Plasma sE-selectin concentration decreased significantly in the PJ group at the end of week 12 compared to baseline (P < 0.001 for treatment effect), and the reduction was significant in comparison with the placebo group (P < 0.05 for treatment effect). There were no significant differences between the two groups in plasma sICAM-1 and sVCAM-1. At the end of the study, compared with the placebo group, NF-κB p65 in PBMC was significantly lower (P < 0.01 for treatment effect) and SIRT1 was significantly higher (P < 0.0001 for treatment effect) in the PJ group.

Conclusion:

This study supports the PJ consumption as a food with potential benefits in individuals with type 2 diabetes as evidenced by improvements in NF-κB and SIRT1 levels in PBMC of study participants.

Introduction

One of the most common causes of death among patients with diabetes mellitus is cardiovascular disease. Therefore, the risk factor management in these patients is vital.¹ It has been thought that chronic inflammation is an important mediator of cardiometabolic dysfunctions.² Increased expression of molecules, such as intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, on the surface of endothelial cells is a critical early event in the development of atherogenesis.³ When circulating levels of these adhesion molecules increased, a proinflammatory state in the vasculature was supposed. A group of nicotinamide adenine dinucleotide-dependent histone deacetylases called sirtuins (silent information regulator proteins) are the major regulator of epigenetic gene silencing. One of the well-defined sirtuins is sirtuin1 (SIRT1) that regulates different cellular functions, including inflammation and glucose–lipid metabolism.⁴ SIRT1 deacetylases wide variety of important genes, including histones and transcription factors such as nuclear factor kappa B (NF-κB). NF-κB controls transcriptional activity of various promoters of genes that mediate inflammation.⁵ SIRT1 physically interacts with the p65 subunit of NF-κB and inhibits transcription by deacetylating p65, leading to the suppression of inflammatory processes.⁶

Pomegranate juice (PJ) is a reach source of polyphenols with the highest antioxidant capacity that have several health benefits.⁷ It has been proved that the beneficial potential of foods rich in polyphenols as part of a health promoting diet cannot be only explained by their antioxidant characteristics and other mechanisms, including their anti-inflammatory properties and interaction with cellular functions, such as binding to nuclear receptors may be involved.⁸ Naturally occurring dietary polyphenols, such as anthocyanins, act as an anti-inflammatory agent through modulating different cellular pathways, such as NF-κB dependent signaling pathways.⁹ In addition, polyphenols have also been shown to activate SIRT1 directly or indirectly in a variety of models.¹⁰ PJ has been reported to improve oxidative stress and systemic inflammation in patients with diabetes.^11

–15 However, molecular mechanism underlying its anti-inflammatory effects has not been investigated in diabetes.

In this study, we evaluated effects of PJ on NF-κB p65 and SIRT1 in peripheral blood mononuclear cells (PBMCs), and on soluble form of ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1), and soluble E-selectin (sE-selectin) in plasma.

Materials and Methods

Study population

Of 289 screened, 50 patients (25 treatment and 25 placebo group) were selected from the outpatient clinic of the Charity Foundation for Special Diseases and Health Center of District 2 of Tehran, Iran. The flowchart of the study was presented in our previous article.¹³ All participants were in the age range of 40 to 65 years and being treated with oral hypoglycemic agents. Participants excluded if they had chronic kidney, liver, or inflammatory diseases; were smoker; were taking estrogen, progesterone, or antioxidant supplements; or were using insulin or anti-inflammatory medications. The study protocol was approved by the Ethics Committee of the National Nutrition and Food Technology Research Institute of Iran. This clinical trial was registered at Iranian Registry of Clinical Trials (IRCT) with number IRCT201206144010N8. http://irct.ir

The study was in adherence with the Declaration of Helsinki. Written, informed consent was obtained from all study participants before initiating the study.

Study design and measurements

This study was a randomized, parallel group, double-blind, placebo-controlled trial. The study participants, after stratification based on sex, were randomly allocated to either PJ or placebo groups by blocked randomization. Randomization was performed by an assistant and the group allocation was blinded for the investigator and participants. Participants in the PJ group consumed 250 mL PJ daily and those in placebo group received a control beverage of similar color and energy content for 12 weeks. PJ was prepared commercially (Alifard, Inc., Tehran, Iran). The placebo was prepared by a special essence and formula from Wonderful Variety, Pom, (Roll International Corporation, Los Angeles, CA). Analysis of the placebo juice using the colorimetric assay verified that it had no polyphenols. Both the PJ and the placebo had similar sugar content and none of the juices had fiber.¹³ The study products were packaged in single-serving bottles.

The study participants were contacted every week to verify if they have any problem in juice consumption and inquire regarding possible side effects. Each patient was provided with a fixed number of the PJ bottles and instructions to return the unused bottles at the end of the study. Based on the number of returned bottles by each patient, their compliance was determined. The participants were excluded from the analysis, if they consumed <90% of the packets, or had changed their medication, or reported severe side effects. Subjects were asked not to change their dietary habits, physical activities, and drug regimens during the study period.

At baseline and at the end of week 12, after a 12- to 14-hr fast, 10 mL blood was collected into heparinized tubes. PBMCs were separated from whole blood by density gradient centrifugation, using Lympholyte-H (Cedarlane, Burlington, Canada), washed with PBS and stored at −70°C. Plasma was prepared by centrifugation, separated into aliquots, and stored at −70°C.

Plasma concentration of sICAM-1 was determined by ELISA kit (Koma Biotech, Seoul, South Korea). Plasma concentrations of sVCAM-1 and sE-selectin were determined by ELISA kits (Cusabio Biotech, Wuhan, China). Intra-assay coefficient of variations for plasma sICAM-1, sVCAM-1, and sE-selectin were 5.8%, 5.8%, and 6.8%, respectively.

PBMCs lysates were prepared and ELISA kits were used to measure total NF-κB p65 (Cell Signaling, MA) and SIRT1 (Abcam, MA) according to the manufacturer's protocol. Intra-assay CVs for NF-κB and SIRT1 were 8.1% and 7%, respectively.

Participants were weighed at baseline and the end of week 12. In addition, the dietary intakes of subjects were assessed using a 3-day dietary recall (2 weekdays and 1 weekend day) at baseline and at the end of week 12. The dietary recalls were analyzed for macronutrients using Nutritionist IV software (N Squared Computing, San Bruno, CA). In addition, intake of total polyphenols was estimated using an online Phenol-Explorer database, which contains mean representative content values for individual polyphenols belonging to four polyphenol classes (flavonoids, phenolic acids, lignans, and stilbenes) and other polyphenols in food products.¹⁶ Physical activity was assessed using the metabolic equivalent of task questionnaire at the beginning and at the end of the trial.¹⁷

Statistical analysis

The minimum sample size estimation for each group was 18 at a power (1-β) of 90% and α = 1.05 for a two-arm parallel study with two-tailed testing to detect a difference of 15 ng/mL in serum E-selectin concentration with a standard deviation of 14 ng/mL, obtained from the study of Asgary.¹⁸

Statistical analysis of data was performed using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL) for windows version 21.0. A chi-squared test was used to compare qualitative variables between the two groups. All quantitative parameters according to the Kolmogorov–Smirnov test had normal distributions, and we used paired t-test to compare parameters within groups. Analysis of covariance was used to compare parameters between groups, entering baseline values and baseline energy and carbohydrate intake as covariate (dietary energy and carbohydrate intake showed significant differences between the two groups at the baseline). The results are expressed as mean ± standard deviation, and differences were considered statistically significant at P < 0.05.

Results

Patient's characteristics

Of the 25 participants who were allocated to each group, 22 successfully completed the study, as presented in the flowchart in our previous article.¹³ In the PJ group, one patient had stomach discomfort, one consumed <90% of juice packets, and the other did not attend at scheduled visit. In placebo group, one had change in treatment plan, one consumed <90% of juice packets, and the other lost last visit. The baseline characteristics of the participants did not differ significantly between the two groups (Table 1). All participants were treated with oral glucose-lowering agents, including insulin release stimulator, metformin, or a combination of these. Based on the participants' reports, lifestyle, and medication, status remained unchanged during the study period. No serious adverse events or side effects were reported.

Table 1.

Baseline Characteristics of the Study Patients

Characteristics	Pomegranate (n = 22)	Placebo (n = 22)	P
Age (years)^a	55 ± 6.7	56.9 ± 6.8	NS
Sex, n (%)
Men	11.0 (50.0)	12.0 (56.0)	NS
Women	11 (50.0)	10.0 (54.5)	NS
Body mass index (kg/m²)^a	29.3 ± 3.9	28.5 ± 4.2	NS
Diabetes duration^a	5.8 ± 3.2	6.5 ± 4.3	NS
Antihypertensive drugs (%) (ACE-I or ARB)	4 (18.2)	6 (27.3)	NS
Antihyperlipidemic drugs (%)	3 (13.6)	4 (18.2)	NS
Oral glucose-lowering agents
Insulin release stimulator (%)	8	6	NS
Metformin (%)	1	2	NS
Both	13	14	NS

Body mass index, age, and duration of diabetes are presented as mean ± SD.

ACE-I, angiotensin converting enzyme inhibitors; ARB, angiotensin receptor blockers; NS, not significant; SD, standard deviation.

There was no significant difference in dietary polyphenol intake between the two groups during the study period, not considering polyphenols contained in PJ (Table 2). Dietary energy and carbohydrate intake showed significant differences between the two groups at the baseline and at the end of the study (Table 2). However, none of the dietary factors changed significantly within each group during the study, and there were no significant differences in intake of other macronutrients. Body weight and body mass index were unchanged throughout the study period in both groups

Table 2.

Dietary Intake and Physical Activity in the Studied Patients

Factors	Groups	Baseline	Week 12	P
Energy (kcal)	Pomegranate	1784 ± 238	1787 ± 233	0.03
Energy (kcal)	Placebo	2028 ± 241	2011 ± 221	0.03
Carbohydrate (grams)	Pomegranate	234 ± 35	266 ± 41	0.04
Carbohydrate (grams)	Placebo	232 ± 27	257 ± 37	0.04
Protein (grams)	Pomegranate	54 ± 12	58 ± 12	NS
Protein (grams)	Placebo	59 ± 10	65 ± 9	NS
Fat (grams)	Pomegranate	73 ± 20	72 ± 19	NS
Fat (grams)	Placebo	84 ± 15	83 ± 19	NS
Fiber (grams)	Pomegranate	15 ± 4	15 ± 3	NS
Fiber (grams)	Placebo	16 ± 3	15 ± 4	NS
Total polyphenol (mg/day)	Pomegranate^a	1964 ± 484	1952 ± 436	NS
Total polyphenol (mg/day)	Placebo	2060 ± 438	2033 ± 494	NS
Flavonoids (mg/day)	Pomegranate	1748 ± 430	1737 ± 388	NS
Flavonoids (mg/day)	Placebo	1834 ± 390	1809 ± 440	NS
Phenolic acids (mg/day)	Pomegranate	156 ± 35	157 ± 39	NS
Phenolic acids (mg/day)	Placebo	166 ± 48	165 ± 35	NS
Lignans, stilbenes, other polyphenols (mg/day)	Pomegranate	59 ± 14	58 ± 15	NS
Lignans, stilbenes, other polyphenols (mg/day)	Placebo	61 ± 13	60 ± 14	NS
Physical activity (MET-h/day)	Pomegranate	37.9 ± 4.6	39.1 ± 4.5	NS
Physical activity (MET-h/day)	Placebo	38.1 ± 3.9	40.2 ± 4.1	NS

All values are presented as mean ± SD. n = 22 for all values.

Polyphenols contained in pomegranate juice that was consumed daily in the pomegranate juice group were not included in the analysis.

MET, metabolic equivalent of task.

Plasma measures

Plasma sICAM-1 reduced significantly in the pomegranate group and the placebo group at the end of week 12 compared to baseline (P < 0.001). However, the difference between two groups was not statistically significant (Table 3). Plasma sE-selectin concentration decreased significantly in the pomegranate (P < 0.001) group compared to baseline, whereas no significant change was observed in the placebo group. The reduction of sE-selectin in the pomegranate group was statistically significant when compared with the placebo group (P < 0.05 for treatment effect; Table 3). No statistically significant differences within or between groups were observed in plasma sICAM-1.

Table 3.

Plasma Concentrations of sICAM-1, sVCAM-1, and sE-Selectin in the Studied Patients

Factors	Baseline	Week 12 ^a	P^b	Change ^a,c	P^d
sICAM-1 (ng/mL)
Placebo	148 ± 12	140 ± 11	0.001	−7.4 ± 6.9	NS
Pomegranate	151 ± 17	138 ± 12	0.001	−12.4 ± 10.9	NS
sVCAM-1 (ng/mL)
Placebo	23 ± 7	20 ± 6	NS	−2.3 ± 7.6	NS
Pomegranate	27 ± 11	31 ± 19	NS	3.3 ± 16.8	NS
sE-selectin (ng/mL)
Placebo	18 ± 8	17 ± 10	NS	−0.3 ± 9.3	0.04
Pomegranate	19 ± 7	13 ± 6	0.001	−6.1 ± 4.6	0.04

All values are presented as mean ± SD. n = 22 for all values.

Between-group variations were analyzed using analysis of covariance adjusted for baseline values, energy, and carbohydrate intake.

vs. baseline.

Changes reflect week 12–baseline values.

vs. the placebo group.

sE-selectin, soluble E-selectin; sICAM-1, soluble intercellular adhesion molecule type 1; sVCAM-1, soluble vascular cell adhesion molecule type 1.

NF-κB p65 and SIRT1

Total NF-κB p65 in PBMC was significantly different between the two groups at baseline (P < 0.01). At the end of the study, the mean difference was significantly lower in the PJ compared with the placebo group, after controlling for baseline values (P < 0.01 for treatment effect).

SIRT1 was significantly different between the two groups at baseline. SIRT1 increased significantly in the pomegranate group at the end of week 12 compared to baseline (P < 0.001). The change from baseline of SIRT1 in the PJ group significantly differed from that of the placebo group (P < 0.0001 for treatment effect) (Table 4).

Table 4.

Sirtuin1 and NF-κB in Peripheral Blood Mononuclear Cells from the Studied Patients

Parameters	Baseline	Week 12 ^a	P^b	Changes ^a,c	P^d
Sirtuin1 (ng/mL)
Placebo	36 ± 15	37 ± 14	NS	0.2 ± 11	0.0001
Pomegranate	26 ± 10^e	43 ± 15	0.001	17 ± 15	0.0001
Total NF-κB p65 (arbitrary unit)
Placebo	1.1 ± 0.5	1.3 ± 0.3	NS	0.2 ± 0.5	0.01
Pomegranate	1.4 ± 0.4^e	1.2 ± 0.2	NS	−0.2 ± 0.4	0.01

All values are presented as mean ± SD. n = 22 for all values.

Between-group variations were analyzed using analysis of covariance adjusted for baseline values, energy, and carbohydrate intake.

vs. baseline.

Changes reflect week 12–baseline values.

vs. the placebo group.

P < 0.01 vs the placebo group.

NF-κB, nuclear factor kappa B.

Discussion

We previously showed that, plasma concentrations of C-reactive protein and interleukin-6 can be reduced in diabetic patients by PJ consumption.¹³ The present study showed that the PJ anti-inflammatory effects may be done by modulating SIRT1- and NF-κB p65-dependent signaling pathways. Active form of NF-κB are nuclear homo- or heterodimeric complexes and the most ubiquitously expressed NF-κB dimer is composed of p50 and p65 subunits, where p65 is the main transcriptional activator.¹⁹ In this study, PJ consumption reduced total p65 concentration in PBMC, and this may have reduced the transcriptional activity of p65. Acetylation of p65 also plays a key role in modulating the transcriptional activity of NF-κB. SIRT1 deacetylases p65 subunit of NF-kB on the lysine 310 residue.⁶ As a result, SIRT1 elevation could suppress NF-κB transcriptional activity. Acetylated NF-κB has not determined in the current study, but it could be hypothesized that the SIRT1 protein concentration increment may have reduced acetylated p65 in PBMC.

The mechanism by which PJ modulated cellular levels of SIRT1 and NF-κB in the current study could be partly attributed to the physiological activities of anthocyanins or other polyphenols contained in the juice. PJ polyphenols, including anthocyanins in the form of water-soluble glycosides, are absorbed in human¹⁸ and kinetic studies have shown that PJ ingestion could lead to plasma elevation of some of these glycosides or metabolites related to them. Delphinidin-glucosides are a group of anthocyanins that can be found in pomegranates.²⁰ In human umbilical vein endothelial cells, delphinidin-3-glucoside has upregulated SIRT1 expression.¹⁹ However, the bioavailability of pomegranate polyphenols should be considered when PJ effect is being evaluated. After ingestion, delphinidin-glucosides can be hydrolyzed by intestinal glucosidases, and the resulting aglycone is subjected to further chemical degradation in the large intestine to form mainly gallic acid.²¹ Oral administration of gallic acid in streptozotocin-induced diabetic rats have significantly inhibited the renal NF-κB activation as well as significantly reduced the serum levels of proinflammatory cytokines.²² In addition, SIRT1 may be involved in the molecular mechanism, by which gallic acid exerts its beneficial effects, since, in a SIRT1 knockdown model, effect of gallic acid on activation of downstream genes was significantly blunted.²³

Cyanidin-glucosides are another type of anthocyanins found in PJ.²⁰ Cyanidin-glucosides of PJ may be degraded by the gut microflora giving rise to the formation of other breakdown metabolites, which could also contribute to the health effect. Cyanidin-glycoside and its metabolite protocatechuic acid have been shown to inhibit NF-κB activation in vivo.²⁴

Ellagitannins are groups of polyphenols abundant in fruit peels of pomegranate. Commercial juices processing of pomegranate fruit led to the extraction of these tannins into the juice. This could account for the higher antioxidant activity of commercial juices compared with the experimental ones.²⁵ Punicalagin is abundant ellagitannins in PJ. It has been thought to be responsible for the high antioxidant activity of PJ.²⁶ However, due to the large size of the molecule, ellagitannins may be assumed to be nonabsorbable and be hydrolyzed in the human gastrointestinal tract, generating ellagic acid which appeared in the plasma after their ingestion, or it may be converted into urolithin A through the action of human colonic microflora.²⁷ The anti-inflammatory properties of PJ also can be related to the ellagic acid and urolithin. Urolithin A inhibited NF-κB translocation to the nucleus upon interleukin-1beta treatment in an in vitro inflammation model in human colonic fibroblasts.²⁸ Gut microbiota modulation apparently could affect the conversion of ellagic acid to urolithin A. Further study is required to investigate whether gut microbiota modulation (i.e., increase of bifidobacteria and lactobacilli), reinforce attenuation of NF-κB activation.

In the current study, PJ has no detectable effects on plasma sICAM-1 and sVCAM-1, whereas it attenuated plasma sE-selectin. Increased plasma levels of these cell adhesion molecules, which are considered as plasma markers of endothelial dysfunction, are associated with cardiovascular and noncardiovascular complications of diabetes.²⁹ E-selectin is only expressed on activated endothelium and regulates adhesive interactions between certain blood cells and endothelium which are one of the initial steps in the pathogenesis of atherosclerosis.³⁰ Endothelial dysfunction in diabetes may be due to increased oxidative stress.³¹ The high antioxidant activity of PJ,⁷ may reduce that. Few studies have evaluated effects of PJ or its polyphenols on these cell adhesion molecules. Pomegranate peel extract and punicalagin have attenuated ICAM-1, but not VCAM-1 release from a human monocytic cell line (THP-1) when exposed to a specific stimulant.³² In contrast to the results of current study, in hypertensive subjects, daily ingestion of 150 mL PJ for 2 weeks reduced serum levels of VCAM-1, whereas elevated those of E-selectin with no significant effect on ICAM-1.³³ This discrepancy may be related to the difference in dose and duration of PJ consumption or study population.

To the best of our knowledge, this randomized, double-blind, placebo-controlled trial was the first study investigating the effect of PJ consumption on SIRT1. Preparing a suitable placebo for PJ, detailed data collection through face-to-face meetings and the stratified blocked randomization design were the most important strengths of the current study. However, limitations of the current study should also be considered when interpreting the results. The main limitation is the small sample size. Concentrations of PJ polyphenols and their metabolites in plasma or PBMC were not measured and this is another limitation of our study.

Conclusions

This study supports the PJ consumption as a food with potential anti-inflammatory effect in individuals with type 2 diabetes. This effect is evidenced by improvements in SIRT1 and NF-κB in PBMC and plasma sE-selectin. Long-term outcome studies are required to test the merits of inclusion of the PJ in the diet of patients with diabetes.

Footnotes

Acknowledgments

This study was supported by the National Nutrition and Food Technology Research Institute of Iran and the Research Institute for Endocrine Sciences. The authors express their gratitude to the subjects for their participation and cooperation in this research and Alifard, Inc., Tehran, Iran for preparing the pomegranate and placebo juices. Special thanks are due to Wonderful Variety, Pom; supplied by Roll International Corporation, Los Angeles, CA, for their placebo formulation.

Author Disclosure Statement

No conflicting financial interests exist.

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