Int J Cardiol 263: 111-117

PMC: PMC5951765

PMID: 29681407

Circulating metabolites of strawberry mediate reductions in vascular inflammation and endothelial dysfunction in db/db mice<a href="#FN3" rid="FN3" class=" fn">1</a>–<a href="#FN6" rid="FN6" class=" fn">5</a>

Background

Cardiovascular disease is 2–4-fold more prevalent in patients with diabetes. Human studies support the cardiovascular benefits of strawberry consumption but the effects of strawberry on diabetic vasculature are unknown. We tested the hypothesis that dietary strawberry supplementation attenuates vascular inflammation and dysfunction in diabetic mice.

Methods

Seven-week-old diabetic db/db mice that consumed standard diet (db/db) or diet supplemented with 2.35% freeze-dried strawberry (db/db+SB) for ten weeks were compared to non-diabetic control mice (db/+). Indices of vascular inflammation and dysfunction were measured. Endothelial cells (ECs) were isolated from the vasculature to determine the influence of strawberry on them. The effect of metabolites of strawberry on endothelial inflammation was determined by incubating mouse aortic ECs (MAECs) with ± 5% serum, obtained from strawberry fed mice (metabolites serum) or standard diet fed mice (control serum) ± 25 mM glucose and 100 μM palmitate.

Results

db/db mice exhibited an increased monocyte binding to vessel, elevated blood pressure, and reduced endothelial-dependent vasorelaxation compared with db/+ mice but each defect was attenuated in db/db+SB mice. The elevation of inflammatory molecules, NOX2 and inhibitor-κB kinase observed in ECs from db/db vs. db/+ mice was suppressed in db/db+SB mice. Glucose and palmitate increased endothelial inflammation in MAECs but were normalized by co-incubation with metabolites serum.

Conclusions

Dietary supplementation of strawberry attenuates indices of vascular inflammation and dysfunction in diabetic db/db mice. The effect of strawberry on vasculature is endothelial-dependent and possibly mediated through their circulating metabolites. Strawberry might complement conventional therapies to improve vascular complications in diabetics.

1. Introduction

Diabetes greatly increases the risk of cardiovascular disease, such as atherosclerosis, which accounts for the largest number of deaths among diabetic patients [1–3]. In diabetes, high glucose induced vascular inflammation and the subsequent endothelial dysfunction plays a major role in the development of vascular disease [2–4]. Hyperglycemia, dyslipidemia, pro-inflammatory cytokines, and vascular adhesion molecules contribute independently and synergistically to vascular dysfunction in diabetes by increasing endothelial cell (EC) inflammation [2–5]. Treating vascular complications associated with diabetes contributes to a heavy economic burden on patients and society. Due to the current epidemic of diabetes, there is a need to identify cost effective adjunct strategies for treating vascular complications associated with this disease.

Human studies support the vascular effects of anthocyanins, one class of flavonoids, widely found in berry fruits [6–12]. Anthocyanins are composed of anthocyanidin (aglycon component of anthocyanins such as pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin) and a sugar moiety. Strawberry is an excellent source of anthocyanins and the most commonly found anthocyanins in strawberries are the glycosidic derivatives of pelargonidin and cyanidin [10]. Recent study shows that consuming 2–3 servings of strawberries per week lowers the risk of myocardial infarction in humans [7]. Strawberry intake was shown to attenuate postprandial inflammation in overweight adults who ingested a high-carbohydrate, moderate-fat meal [13]. Further, intake of strawberries reduces vascular adhesion molecules in humans with cardiovascular risk factors [11, 12]. However, the effects of strawberry on endothelial dysfunction in general and diabetes in particular are unknown. Anthocyanins are extensively metabolized in humans by digestive enzymes and intestinal microbiota, suggesting the vascular effects could be mediated by their circulating metabolites [6, 14–17]. Indeed, data from our laboratory and others suggest strongly that anthocyanin metabolites and not the parent anthocyanins might be the main cause of some of the benefits of the ingested parent compound [6, 18, 19].

In the present study, we investigated whether dietary supplementation of strawberries, at a nutritional dose, ameliorates vascular inflammation and dysfunction in diabetes. Further, we examined whether the effect of strawberry on vasculature is endothelial-dependent and the role of metabolites in exerting the vascular effects of strawberries. Specifically, we hypothesized that strawberry ameliorates vascular inflammation and dysfunction in diabetic mice and that the endothelial specific vascular beneficial effects of strawberry are mediated through their circulating metabolites.

2. Materials and methods

2.1. Materials

Materials are available in the online supporting material.

2.2. Pre-clinical model of type 2 diabetes

Six-week-old male diabetic db/db mice with a C57BLKS/J background (db/db; B6.Cg-m^+/+Lepr^db) and control db/+ mice with the same background (Stock no. 000642) were obtained from the Jackson Laboratories (Bar Harbor, ME). db/db mice is a widely-used type 2 diabetic animal model that spontaneously develops endothelial dysfunction and vascular complications [4, 20–22]. We have previously demonstrated an enhanced endothelial inflammation in db/db mice [4, 20]. The mice were provided free access to a AIN 93 G rodent diet (Dyets Inc, Bethlehem, PA) with corn oil substituted for soy bean oil [4], and acclimated for a week before experiments were performed. Mice were exposed to experimental conditions beginning at seven weeks of age. Animals were maintained under artificial light in a 12-hour light/dark cycle, 23 ± 1° C, and 45 ± 5% humidity and were held under humane conditions. The Institutional Animal Care and Use Committee at the University of Utah approved animal experimental protocols.

2.3. Standard diet and strawberry supplemented diet

The freeze-dried strawberry powder was provided by the FutureCeuticals (Momence, IL). The customized pelleted diets prepared as shown in Supplementary Table 1 were supplied by Dyets Inc. (Bethlehem, PA). The strawberry supplemented diet was adjusted to compensate for the additional sugars and fiber provided by the freeze-dried strawberry. The amount of freeze-dried strawberry powder used in this study was calculated based on the extrapolation of doses from animals to humans by normalization to body surface area [23]. The nutritional dose of freeze-dried strawberry powder was based on average human consumption. 2.35% Freeze-dried strawberry powder in diet (w/w) is equivalent to two human servings of fresh strawberries (~160 g strawberries) per day [24].

2.4. Experimental groups

After 1 week of acclimation, db/db mice (seven-week-old) were divided into two groups and received standard diet (db/db) (n=20) or 2.35% freeze-dried strawberry supplemented diet (db/db+SB) (n=16) for 10 weeks. db/+ mice (seven-week-old) received standard diet (db/+) (n=20) for 10 weeks. The markers of inflammation and relevant endpoints were assessed at the organism level, organ level, and cellular level (Supplementary Fig. 1A).

2.5. Measurement of metabolic variables, blood pressure and collection of tissue samples

Blood glucose, serum lipids, glucose tolerance, insulin tolerance and blood pressure were measured as we have described [4, 20, 25] and detailed in the online supplement.

2.6. Measurement of vascular inflammation

The effect of strawberry supplementation on vascular inflammation in diabetic mice was assessed by measuring the binding of monocytes to the aortic vessel, determining the expression of inflammatory markers [chemokines such as monocyte chemotactic protein-1 (MCP-1)/JE and KC, and adhesion molecules such as vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-Selectin] in aortic vessels, and measuring circulating chemokines as we have described [4, 19, 26] and detailed in the online supplement.

2.7. Measurement of vascular function

Vascular function was assessed in two mesenteric artery segments using isometric tension techniques as we have described [25, 27–29] and detailed in the online supplement.

2.8. Isolation of ECs from carotid artery and assessment of endothelial effects of strawberry

To assess the influence of strawberry metabolites on endothelial cells, we further investigated vascular ECs for markers of inflammation. ECs were isolated from carotid arteries as we have described [30]. Briefly, carotid arteries were excised and perfused with QIAzol reagent. The vessel effluent containing the intimal fraction contains the carotid artery ECs (CAECs). The remaining components of the vessel contains media and adventitia. The purity of CAECs in the vessel effluent was confirmed by measuring platelet endothelial cell adhesion molecule-1 (PECAM-1) which is specific to ECs and smooth muscle actin (SMA) which is specific for smooth muscle cells. The mRNA expression of inflammatory and adhesion molecules, NADPH oxidases (NOXs), nuclear factor-κB (NFκB) signaling markers, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-α (TNF-α), and nuclear factor erythroid 2 - related factor (Nrf2) were measured by qPCR [18].

2.9. Cell culture

Mouse aortic endothelial cells (MAECs) and mouse monocytic WEHI78/24 cells were cultured as described in the online supplement.

2.10. Measuring the effects of serum of strawberry-fed mice on endothelial inflammation

To assess whether the vascular effects of strawberry are mediated through their circulating metabolites we used serum derived from strawberry fed mice. Serum was collected at the fed state from non-diabetic control mice (db/+ mice) supplemented with standard diet (control serum) or 2.35% freeze-dried strawberry diet (metabolites serum) for 10 weeks. MAECs were grown to confluence in EC complete medium. After serum starvation for 12 h, MAECs (passage 4–5) were treated with or without 5% serum derived from strawberry fed mice (metabolites serum) or standard diet fed mice (control serum) and with or without 25 mM glucose (HG) and 100 μM palmitate-BSA (Pal) (or) mannose and BSA (vehicle) for 3 days (Supplementary Fig. 1B). Palmitate (C16:0) (Sigma, MO) was coupled to fatty acid-free BSA in the ratio of 2 M palmitate to 1 M BSA [25]. HG+Pal is an established model to induce endothelial inflammation and the doses of glucose/palmitate were chosen based on a previous study indicating its ability to evoke endothelial inflammation in EC [31]. Upon completing the respective treatments, the following were assessed: WEHI78/24 mouse monocyte binding to MAEC and secretion of inflammatory chemokines in the medium.

2.11. Data Analysis

Comparison of one time point among groups was made using one-way ANOVA. All data points at all times in glucose and insulin tolerance tests were compared using repeated measures ANOVA. To analyze vascular function, comparison of multiple time points among groups was made using two-way repeated-measures ANOVA. Significant differences among means were identified by the Tukey post hoc test. Data were analyzed with the use of SPSS (Version 25; IBM) or Prism (Version 7.0; GraphPad). All data were expressed as mean ± SEM, and P < 0.05 was considered statistically significant.

2.1. Materials

Materials are available in the online supporting material.

2.2. Pre-clinical model of type 2 diabetes

2.3. Standard diet and strawberry supplemented diet

2.4. Experimental groups

2.5. Measurement of metabolic variables, blood pressure and collection of tissue samples

Blood glucose, serum lipids, glucose tolerance, insulin tolerance and blood pressure were measured as we have described [4, 20, 25] and detailed in the online supplement.

2.6. Measurement of vascular inflammation

2.7. Measurement of vascular function

Vascular function was assessed in two mesenteric artery segments using isometric tension techniques as we have described [25, 27–29] and detailed in the online supplement.

2.8. Isolation of ECs from carotid artery and assessment of endothelial effects of strawberry

2.9. Cell culture

Mouse aortic endothelial cells (MAECs) and mouse monocytic WEHI78/24 cells were cultured as described in the online supplement.

2.10. Measuring the effects of serum of strawberry-fed mice on endothelial inflammation

2.11. Data Analysis

3. Results

3.1. Strawberry supplementation does not alter the systemic metabolic milieu in db/db mice

Body weight, food intake, fasting and non-fasting blood glucose, and serum cholesterol and triglycerides were increased in db/db mice vs. db/+ mice, but were not altered in db/db+SB vs. db/db mice (Supplementary Table 2). Likewise, strawberry supplementation did not alter the disruption of peripheral glucose homeostasis observed in db/db mice compared with db/+ mice (Supplementary Fig. 2A, B).

3.2. Strawberry supplementation reduces vascular inflammation and modestly improves lipid peroxidation in db/db mice

There was an increase in binding of mouse monocytic WEHI 78/24 cells to the aortic vessel isolated from db/db mice compared with db/+ mice (Fig. 1A). However, strawberry consumption reduced the binding of monocytes to aortic vessels in db/db+SB vs. db/+ mice (Fig. 1A). The aortic vessels isolated from db/db mice exhibited a significant increase in the mRNA expression of MCP-1/JE, KC, and VCAM-1 compared with db/+ mice (Fig. 1B, C). MCP-1/JE and KC are the mouse ortholog of MCP-1 and IL-8 respectively [4]. However, the expression of MCP-1/JE, KC, and VCAM-1 were significantly reduced in db/db+SB vs. db/db mice (Fig. 1B, C). Consistently, MCP-1/JE and KC were significantly increased in the serum of db/db mice compared with db/+ mice but db/db+SB mice exhibited decreased levels of these serum chemokines (Fig. 1D, E). The lipid hydroperoxidation, as measured by the concentration of serum lipid hydroperoxides was increased in db/db vs. db/+ mice and strawberry supplementation tended to decrease (p = 0.06) lipid hydroperoxides in db/db+SB mice (Fig. 1F).

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Fig. 1

Monocyte binding (A), inflammatory chemokines (B) and adhesion molecules (C) in aortic vessels, serum MCP1/JE (D), serum KC (E) and serum hydroperoxides (F) of db/+, db/db and db/db+SB mice treated for 10 wk. Values are mean ± SEM, n=5–6. Comparison among groups was made using one-way ANOVA and Tukey post hoc tests were performed when significant main effects were obtained. Labelled means without a common letter differ, P < 0.05. db/+, Standard diet fed control mice; db/db, standard diet fed diabetic mice; db/db+SB, strawberry fed diabetic mice; E-sel, E-selectin; ICAM-1, intercellular adhesion molecule-1; MCP1, monocyte chemoattractant protein-1; VCAM-1, vascular cell adhesion molecule-1.

3.3. Blood pressure was lower and endothelial vasorelaxation was improved in the strawberry supplemented db/db mice

Arterial pressure (systolic and diastolic) increased in db/db vs. db/+ mice (Supplementary Table 2). However, the severity of hypertension was lower in db/db+SB vs. db/db mice. Relaxation to acetylcholine in mesenteric arteries precontracted with phenylephrine was impaired in db/db mice compared with db/+ mice (Fig. 2A). Of note, the severity of impairment was lessened in db/db+SB vs. db/db mice (Fig. 2A). Importantly, endothelium-independent vasorelaxation was similar among groups (Fig. 2B), suggesting strongly that an endothelium-specific defect that existed in db/db mice was ameliorated by strawberry supplementation. The receptor and non-receptor mediated vasocontraction were similar among groups (Fig. 2C, D).

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Fig. 2

Endothelium-dependent vasorelaxation (A), endothelium-independent vasorelaxation (B), receptor-mediated vasocontraction (C), and non-receptor mediated vasocontraction (D) in db/+, db/db and db/db+SB mice treated for 10 wk. Values are mean ± SEM, n=5–6. Comparison of multiple time points among groups was made using two-way repeated-measures ANOVA and Tukey post hoc tests were performed when significant main effects were obtained. Labelled means without a common letter differ, P < 0.05. db/+, Standard diet fed control mice; db/db, standard diet fed diabetic mice; db/db+SB, strawberry fed diabetic mice.

3.4. Strawberry supplementation reduces endothelial inflammatory molecules in db/db mice indicating the endothelial effects of strawberry bioactives

The endothelial effect of strawberry was assessed by measuring inflammatory molecules in CAECs isolated from experimental mice. The purity of CAECs in intimal fractions was confirmed by the presence of PECAM-1 and absence of SMA in the intimal fraction (Fig. 3A, B). CAECs isolated from db/db mice exhibited a significant increase in mRNA expression of MCP1/JE, KC, ICAM1 and VCAM1 as compared with the CAECs from db/+ mice (Fig. 3C, D). However, the mRNA expression of MCP1/JE, KC, and VCAM1 was significantly reduced in CAECs from db/db+SB vs. db/db mice (Fig. 3C, D). This indicates an endothelial specific anti-inflammatory effect of strawberry bioactives.

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Fig. 3

(A) PECAM-1 and SMA in intimal fraction. (B) PECAM-1 in intimal fraction and media/adventitia. Expression of inflammatory chemokines (C), adhesion molecules (D), NFκB signaling markers (E), and NADPH oxidases (NOXs) (F) in CAECs isolated from db/+, db/db and db/db+SB mice treated for 10 wk. Values are mean ± SEM, n=5–6. Comparison among groups was made using one-way ANOVA and Tukey post hoc tests were performed when significant main effects were obtained. Labelled means without a common letter differ, P < 0.05. CAECs, carotid artery endothelial cells; db/+, Standard diet fed control mice; db/db, standard diet fed diabetic mice; db/db+SB, strawberry fed diabetic mice; E-Sel, E-selectin; ICAM-1, intercellular adhesion molecule-1; IκKβ, inhibitor κB kinase, PECAM-1, platelet endothelial cell adhesion molecule-1; SMA, smooth muscle actin; VCAM-1, vascular adhesion molecule-1.

3.5. Strawberry supplementation reduces the expression of inhibitor κB kinase (IκKβ) and NOX2 in db/db mice

We further assessed whether the effect of strawberry on ECs is mediated through NFκB, NOXs, eNOS or Nrf2. CAECs isolated from db/db mice exhibited an increased expression of IκKβ, IκBα, NOX2, and NOX4 compared with CAECs from db/+ mice (Fig. 3E, F). Interestingly, the expression of IκKβ and NOX2 were reduced in CAECs from db/db+SB vs. db/db mice (Fig. 3E, F). Further, the endothelial expression of IκBα (p = 0.06) and NOX4 (p = 0.06) were modestly reduced in CAECs from db/db+SB vs. db/db mice. The expression of eNOS, TNF-α, and Nrf2 were similar among groups (Supplementary Fig. 3).

3.6. Strawberry metabolites suppress endothelial inflammation in MAECs exposed to high glucose and palmitate

HG+Pal treatment significantly increased the binding of mouse monocytic WEHI78/24 cells to MAEC which was associated with a significant increase in the secretion of MCP-1/JE and KC when compared with vehicle treated MAECs (Fig. 4A, B, C). However, co-treatment with serum obtained from strawberry fed mice significantly reduced monocyte binding to MAEC and reduced the secretion of the KC compared with control serum treated MAECs (Fig. 4A, C). This indicates the possible role of circulating metabolites in exerting the endothelial/vascular effects of strawberry.

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Fig. 4

Monocytes binding to MAECs (A), and secretion of MCP1/JE (B) and KC (C) in MAECs treated with ± HG/Pal or Man/BSA ± metabolites serum or control serum for 3 days. Values are mean ± SEM, n=5. Comparison among groups was made using one-way ANOVA and Tukey post hoc tests were performed when significant main effects were obtained. Labelled means without a common letter differ, P < 0.05. BSA: bovine serum albumin; HG, High glucose; MAECs, mouse arotic endothelial cells; Man, Mannose; MCP1 – monocyte chemoattractant protein-1; Pal, palmitate-BSA.

3.1. Strawberry supplementation does not alter the systemic metabolic milieu in db/db mice

3.2. Strawberry supplementation reduces vascular inflammation and modestly improves lipid peroxidation in db/db mice

Fig. 1

3.3. Blood pressure was lower and endothelial vasorelaxation was improved in the strawberry supplemented db/db mice

Fig. 2

3.4. Strawberry supplementation reduces endothelial inflammatory molecules in db/db mice indicating the endothelial effects of strawberry bioactives

Fig. 3

3.5. Strawberry supplementation reduces the expression of inhibitor κB kinase (IκKβ) and NOX2 in db/db mice

3.6. Strawberry metabolites suppress endothelial inflammation in MAECs exposed to high glucose and palmitate

Fig. 4

4. Discussion

Epidemiological and clinical studies support the cardiovascular benefits of strawberry consumption [7, 10]. We investigated the hypothesis that dietary strawberry at a nutritional dose ameliorates vascular complications in db/db mice. Our results indicate that dietary supplementation of strawberry reduces vascular inflammation and endothelial dysfunction that otherwise was displayed by db/db mice. Further, the vascular effects of strawberry appeared to be endothelial specific and likely mediated through their circulating metabolites.

The influence of strawberry supplementation on endothelial inflammation and dysfunction was assessed because both contribute importantly to the pathogenesis of atherosclerosis. The binding of monocytes to the aortic vessel followed by their transmigration into the subendothelial space play a major role in the development of atherosclerosis [9]. Inflammatory chemokines and adhesion molecules are involved in monocyte rolling, and their interaction with endothelium [9, 18]. In the present study, mouse monocytic WEHI 78/24 cells had significantly higher binding to the aortic vessel isolated from db/db mice vs. db/+ mice. This was accompanied by the increased expression of MCP1/JE, KC, ICAM1, and VCAM1 in the aortic vessels of db/db mice compared with control mice as reported in previous studies [32, 33]. Strawberry supplementation suppressed MCP-1/JE, KC, and VCAM1 in db/db+SB mice which is associated with reduced binding of monocytic WEHI78/24 cells to the diabetic vasculature. This is consistent with a previous study that showed strawberry supplementation reduces circulating ICAM1 and VCAM1 in humans with cardiovascular risk factors [11, 12]. Further, strawberry supplementation reduced the serum MCP-1/JE and KC in db/db+SB compared with db/db mice. Our data indicate that the effects of strawberry on inflammatory molecules are regulated both at the transcriptional and translational levels.

Endothelial dysfunction plays a pivotal role in the development of vascular complications and atherosclerosis in type 2 diabetes [22, 33]. Evidence shows that endothelial dysfunction associated with impaired endothelium-dependent vasodilation in resistance arteries contributes to increased blood pressure in db/db mice [34, 35]. In our study, db/db mice exhibited an increase in blood pressure compared with control db/+ mice as we have reported previously [4, 20]. However, few studies reported normal or mild elevation of blood pressure values in db/db mice as compared with control C57BLKS/6J mice [36, 37]. This discrepancy could be due to the different control animals used in these studies. In the present study, arterial hypertension observed in db/db mice was lowered by dietary supplementation of strawberry. Our experimental design included parallel assessment of vascular function in mesenteric arteries from all groups, because these resistance-sized vessels are known to contribute importantly to peripheral resistance and blood pressure [38]. Consistent with previous studies, vasorelaxation evoked by acetylcholine (i.e., endothelium-dependent) but not sodium nitroprusside (endothelium-independent) was impaired in db/db mice compared with control mice [34, 35]. As hypothesized, the severity of dysfunction observed in db/db mice was attenuated in db/db+SB mice. Hence, it is not unreasonable to speculate that dietary strawberry might have improved arterial pressure in db/db mice secondary to improved endothelial function. However, we cannot exclude the possibility that the bioactives of strawberry may act on multiple targets to exert the vascular beneficial effects of strawberry.

Next we sought to determine a possible cause for the endothelial-specific effect of strawberry. In this regard, elevated mRNA expression of MCP1/JE, KC, ICAM1, and VCAM1 observed in CAECs from db/db mice was attenuated in db/db+SB mice. We interpret these data to indicate that strawberry supplementation has a direct effect on endothelial cells. Taken together, our study is the first to provide the evidence for the benefits of strawberry on vascular inflammation at the organism level (mice), tissue level (vessel), and cellular level (ECs).

Dietary supplementation of strawberry did not affect metabolic parameters such as body weight, food intake, blood glucose, serum lipids, glucose or insulin tolerance in db/db+SB mice. This indicates that the observed vascular effects of strawberry are not mediated through the improvement in these metabolic parameters and are not likely to be due to any secondary effect.

Anthocyanins are extensively metabolized in humans suggesting the vascular benefits of strawberry might be mediated by their circulating metabolites [6, 14–17]. Indeed, data from our laboratory and others suggest that anthocyanin metabolites might be the principle actors in some of the benefits of the ingested parent compound [6, 18, 19]. To further explore this, we assessed whether the vascular and endothelial effects are mediated through the metabolites of strawberry. In our study, serum containing strawberry metabolites suppressed HG+Pal-induced monocyte binding to MAEC and reduced the secretion of inflammatory KC by MAECs. A recent study showed that 21 polyphenolic metabolites appear in the plasma following the consumption of strawberries in humans [17]. Pelargonidin-3-O-glucuronide (P3G) is the major metabolite of the most abundant anthocyanin pelargonidin-3-O-glucoside [16, 17]. We speculate that the endothelial benefits could be due to these strawberry metabolites present in the serum. Further, we used 5% serum in our in vitro study in which strawberry metabolites were diluted 20x their normal concentration and thus the results might have been even more significant in vivo.

We also determined the possible mechanisms involved in the vascular effects of strawberry. NOXs are a family of reactive oxygen species (ROS) generating enzymes and the effects of NOX1, NOX2 and NOX4 on vasculature are well documented [39, 40]. NOX-derived ROS play a major role in the maintenance of normal physiological vascular function as well as vascular pathology such as the development of endothelial dysfunction, inflammation, and atherosclerosis [40]. Indeed, an increased mRNA expression of NOX2, gp91phox and p22phox subunits of NOX was reported in diabetic vasculature [32, 33, 39]. Consistent with these studies, in our study the mRNA expression of NOX2 and NOX4 were greatly increased in CAECs isolated from db/db mice vs. db/+ mice. Strawberry supplementation significantly reduced NOX2 and modestly reduced NOX4 in db/db mice indicating the effect of strawberry could be mediated through NOXs. In addition, strawberry supplementation modestly reduced serum lipid hydroperoxides in db/db mice. NFκB is one of the major targets of NOX derived ROS and NOXs activate NFκB signaling in diabetic vasculature [20, 41, 42]. p50/p65 is the most abundant form of NFκB. IKKβ activates the nuclear translocation of p50/p65 by degrading the inhibitor IκBα [20]. In the nucleus, p50/p65 binds to the promoters of NFκB-dependent inflammatory genes and upregulates the genes involved in atherosclerosis [20]. High glucose increases ROS which activates p65 nuclear translocation in ECs and aortic vessels that participate in diabetes-caused vascular complications [20, 43]. In our study, CAECs from db/db mice exhibited an increased expression of IKKβ and IκBα. Increased IKKβ expression indicates the activation of NFκB signaling and increased IκBα expression could be an adaptive response to this activation. Strawberry supplementation significantly reduced the expression of IKKβ and modestly reduced IκBα in CAECs of db/db+SB mice. These data indicate that strawberry likely attenuates NFκB signaling by suppressing NOX in db/db+SB mice.

4.1. Limitations

In our study, we assessed the mRNA expression in CAECs and aortic vessels, determined the vascular inflammation in aortic vessels, and measured the vascular function in segments of mesenteric artery. Studies indicate that the gene expression is differentially regulated throughout the vasculature because of the heterogeneity in the structure and function of the endothelium [44, 45]. Hence, the effect of strawberry on vasculature could depend on their anatomical location and we acknowledge this limitation. However, it is important to note that the comparisons between db/+, db/db, and db/db+SB mice in this study were performed in the same segment of the arterial tree. Further, our study indicates that the vascular benefits of strawberries could be due to strawberry metabolites present in the serum. However, we do not know with certainty as we did not measure the metabolites in the serum. Future research is warranted to better understand and validate the bioactivities of strawberries.

4.2. Conclusions

Our findings indicate that dietary supplementation of strawberry ameliorates endothelial dysfunction, improves vascular inflammation, and reduces endothelial inflammatory and adhesion molecules in db/db mice. Further, we provide robust evidence that the effect of strawberry on diabetic vasculature is endothelial specific and is possibly mediated via circulating metabolites of anthocyanins. Of note, the vascular benefits of strawberry were achieved at a nutritional dose, i.e., one that is equivalent to 160 g of strawberries. This is an important consideration when contemplating the translational relevance of findings from diabetic mice to patients with debilitating disease. Further, our results promote further exploration into the effect of strawberry on inflammatory and atherogenic pathways and these studies are ongoing. In conclusion, our study provides strong proof of concept for further considering strawberry as an adjunct therapy to preventing, delaying the onset, or reversing vascular complications associated with diabetes.

4.1. Limitations

4.2. Conclusions

Supplementary Material

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Acknowledgments

C.P., and P.V.A.B. designed research; C.P., D.B., B.R.C., S.G., C.D., J.E.M., J.C., J.K., J.D.S., and P.V.A.B. conducted research; C.P., J.D.S., and P.V.A.B. analyzed data; and C.P., J.D.S. and P.V.A.B. wrote the paper. P.V.A.B. had primary responsibility for final content. All authors read and approved the final manuscript.

^{Department of Nutrition and Integrative Physiology, College of Health, University of Utah, Salt Lake City, Utah 84112, USA}

^{Division of Endocrinology, Metabolism, and Diabetes; and Molecular Medicine Program, University of Utah, Salt Lake City, UT 84112, USA}

^{Corresponding author: Pon Velayutham Anandh Babu, Department of Nutrition and Integrative Physiology, College of Health, University of Utah, Salt Lake City, Utah 84112. Tel: +1-801-581-8376, Fax: +1-801-585-3874,}ude.hatu@mahtuyalev.hdnana

^{Current address for J.K.: Department of Physical Education, Gyeongsang National University, South Korea.}

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Abstract

Background

Methods

Results

Conclusions

Keywords: strawberry, vascular inflammation, endothelial dysfunction, diabetes, metabolites

Abstract

Highlights

Footnotes

^{Supplementary Methods}, Tables and Figures are available from the “Online Supporting Material”

^{Abbreviations: BSA, bovine serum albumin; CAECs, carotid artery endothelial cells; DAF, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate;}db/+, non-diabetic control mice; db/db, diabetic mice; db/++SB, strawberry fed control mice; db/db+SB, strawberry fed diabetic mice; DCFDA, 2′,7′-Dichlorofluorescein diacetate; eNOS, endothelial nitric oxide synthase; HG, 25 mM glucose; IL-8, interleukin-8; ICAM-1, intercellular adhesion molecule-1; IκKβ, inhibitor κB kinase; MAECs, mouse aortic endothelial cells; MCP1, monocyte chemoattractant protein-1; NFκB, nuclear factor κB; L-NAME, L-N^{-Nitroarginine methyl ester; NO, nitric oxide; NOX, NADPH Oxidases; Pal, 100 μM palmitate-BSA; ROS, reactive oxygen species; TNF-α, Tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1.}

^{Supported by research funds from the University of Utah research start-up fund, University of Utah Seed Grant, and College of Health Pilot Grant (to P.V.A.B.); the University of Utah Undergraduate Research Opportunities Program award (to B.C., S.G., J.E.M.); Native American Research Internship (to C.D.); American Heart Association (AHA:16GRNT31050004) and National Institute of Health (NIH: R03AGO52848) (to J.D.S.).}

^{All above authors takes responsibility of all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. Authors declare no potential conflict of interest.}

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Footnotes

References

1. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, et al Heart disease and stroke statistics--2013 update: a report from the American Heart Association. Circulation. 2013;127:e6–e245.[Google Scholar]
2. Chistiakov DA, Orekhov AN, Bobryshev YVEndothelial Barrier and Its Abnormalities in Cardiovascular Disease. Front Physiol. 2015;6:365.[Google Scholar]
3. Tabit CE, Chung WB, Hamburg NM, Vita JAEndothelial dysfunction in diabetes mellitus: molecular mechanisms and clinical implications. Rev Endocr Metab Disord. 2010;11:61–74.[Google Scholar]
4. Babu PV, Si H, Fu Z, Zhen W, Liu DGenistein prevents hyperglycemia-induced monocyte adhesion to human aortic endothelial cells through preservation of the cAMP signaling pathway and ameliorates vascular inflammation in obese diabetic mice. J Nutr. 2012;142:724–30.[Google Scholar]
5. Symons JD, Abel EDLipotoxicity contributes to endothelial dysfunction: A focus on the contribution from ceramide. Rev Endocr Metabol Disord. 2013;14:59–68.[Google Scholar]
6. Rodriguez-Mateos A, Rendeiro C, Bergillos-Meca T, Tabatabaee S, George TW, Heiss C, et al Intake and time dependence of blueberry flavonoid-induced improvements in vascular function: a randomized, controlled, double-blind, crossover intervention study with mechanistic insights into biological activity. Am J Clin Nutr. 2013;98:1179–91.[PubMed][Google Scholar]
7. Cassidy A, Mukamal KJ, Liu L, Franz M, Eliassen AH, Rimm EBHigh anthocyanin intake is associated with a reduced risk of myocardial infarction in young and middle-aged women. Circulation. 2013;127:188–96.[Google Scholar]
8. Basu A, Du M, Leyva MJ, Sanchez K, Betts NM, Wu M, et al Blueberries decrease cardiovascular risk factors in obese men and women with metabolic syndrome. J Nutr. 2010;140:1582–7.[Google Scholar]
9. Cutler BR, Petersen C, Anandh Babu PVMechanistic insights into the vascular effects of blueberries: Evidence from recent studies. Mol Nutr Food Res. 2017;61:1600271.[PubMed][Google Scholar]
10. Giampieri F, Forbes-Hernandez TY, Gasparrini M, Alvarez-Suarez JM, Afrin S, Bompadre S, et al Strawberry as a health promoter: an evidence based review. Food Func. 2015;6:1386–98.[PubMed][Google Scholar]
11. Djurica D, Holt RR, Ren J, Shindel AW, Hackman RM, Keen CLEffects of a dietary strawberry powder on parameters of vascular health in adolescent males. Br J Nutr. 2016;116:639–47.[PubMed][Google Scholar]
12. Basu A, Fu DX, Wilkinson M, Simmons B, Wu M, Betts NM, et al Strawberries decrease atherosclerotic markers in subjects with metabolic syndrome. Nutr Res. 2010;30:462–9.[Google Scholar]
13. Edirisinghe I, Banaszewski K, Cappozzo J, Sandhya K, Ellis CL, Tadapaneni R, et al Strawberry anthocyanin and its association with postprandial inflammation and insulin. Br J Nutr. 2011;106:913–22.[PubMed][Google Scholar]
14. de Ferrars RM, Czank C, Zhang Q, Botting NP, Kroon PA, Cassidy A, et al The pharmacokinetics of anthocyanins and their metabolites in humans. Br J Pharmacol. 2014;171:3268–82.[Google Scholar]
15. de Ferrars RM, Cassidy A, Curtis P, Kay CDPhenolic metabolites of anthocyanins following a dietary intervention study in post-menopausal women. Mol Nutr Food Res. 2014;58:490–502.[PubMed][Google Scholar]
16. Mullen W, Edwards CA, Serafini M, Crozier ABioavailability of pelargonidin-3-O-glucoside and its metabolites in humans following the ingestion of strawberries with and without cream. J Agric Food Chem. 2008;56:713–9.[PubMed][Google Scholar]
17. Sandhu AK, Miller MG, Thangthaeng N, Scott TM, Shukitt-Hale B, Edirisinghe I, et al Metabolic fate of strawberry polyphenols after chronic intake in healthy older adults. Food Func. 2018;9:96–106.[PubMed][Google Scholar]
18. Bharat D, Cavalcanti RRM, Petersen C, Begaye N, Cutler BR, Costa MMA, et al Blueberry Metabolites Attenuate Lipotoxicity-Induced Endothelial Dysfunction. Mol Nutr Food Res. 2018;62:1700601.[PubMed][Google Scholar]
19. Cutler B, Gholami S, Chua JS, Kuberan B, Anandh Babu PVBlueberry metabolites restore cell surface glycosaminoglycans and attenuate endothelial inflammation in diabetic human aortic endothelial cells. International journal of cardiology. 2018 in press. [Google Scholar]
20. Babu PV, Si H, Liu DEpigallocatechin gallate reduces vascular inflammation in db/db mice possibly through an NF-kappaB-mediated mechanism. Mol Nutr Food Res. 2012;56:1424–32.[Google Scholar]
21. Zhong JC, Yu XY, Huang Y, Yung LM, Lau CW, Lin SGApelin modulates aortic vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic mice. Cardiovas Res. 2007;74:388–95.[PubMed][Google Scholar]
22. Li Q, Atochin D, Kashiwagi S, Earle J, Wang A, Mandeville E, et al Deficient eNOS phosphorylation is a mechanism for diabetic vascular dysfunction contributing to increased stroke size. Stroke. 2013;44:3183–8.[Google Scholar]
23. Nair AB, Jacob SA simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. 2016;7:27–31.[Google Scholar]
24. Parelman MA, Storms DH, Kirschke CP, Huang L, Zunino SJDietary strawberry powder reduces blood glucose concentrations in obese and lean C57BL/6 mice, and selectively lowers plasma C-reactive protein in lean mice. Br J Nutr. 2012;108:1789–99.[PubMed][Google Scholar]
25. Bharath LP, Ruan T, Li Y, Ravindran A, Wan X, Nhan JK, et al Ceramide-Initiated Protein Phosphatase 2A Activation Contributes to Arterial Dysfunction In Vivo. Diabetes. 2015;64:3914–26.[Google Scholar]
26. Nallasamy P, Si H, Babu PV, Pan D, Fu Y, Brooke EA, et al Sulforaphane reduces vascular inflammation in mice and prevents TNF-alpha-induced monocyte adhesion to primary endothelial cells through interfering with the NF-kappaB pathway. J Nutr Biochem. 2014;25:824–33.[Google Scholar]
27. Zhang QJ, Holland WL, Wilson L, Tanner JM, Kearns D, Cahoon JM, et al Ceramide mediates vascular dysfunction in diet-induced obesity by PP2A-mediated dephosphorylation of the eNOS-Akt complex. Diabetes. 2012;61:1848–59.[Google Scholar]
28. Carlstrom J, Symons JD, Wu TC, Bruno RS, Litwin SE, Jalili TA quercetin supplemented diet does not prevent cardiovascular complications in spontaneously hypertensive rats. J Nutr. 2007;137:628–33.[PubMed][Google Scholar]
29. Symons JD, McMillin SL, Riehle C, Tanner J, Palionyte M, Hillas E, et al Contribution of insulin and Akt1 signaling to endothelial nitric oxide synthase in the regulation of endothelial function and blood pressure. Circ Res. 2009;104:1085–94.[Google Scholar]
30. Bharath LP, Cho JM, Park SK, Ruan T, Li Y, Mueller R, et al Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase. Arterioscler Thromb Vasc Biol. 2017[Google Scholar]
31. Durpes MC, Morin C, Paquin-Veillet J, Beland R, Pare M, Guimond MO, et al PKC-beta activation inhibits IL-18-binding protein causing endothelial dysfunction and diabetic atherosclerosis. Cardiovas Res. 2015;106:303–13.[Google Scholar]
32. Shen M, Sun D, Li W, Liu B, Wang S, Zhang Z, et al The synergistic effect of valsartan and LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] on vascular oxidative stress and inflammation in type 2 diabetic mice. Exp Diab Res. 2012;2012:146194.[Google Scholar]
33. Lee S, Zhang H, Chen J, Dellsperger KC, Hill MA, Zhang CAdiponectin abates diabetes -induced endothelial dysfunction by suppressing oxidative stress, adhesion molecules, and inflammation in type 2 diabetic mice. Am J Physiol. 2012;303:H106–15.[Google Scholar]
34. Su W, Guo Z, Randall DC, Cassis L, Brown DR, Gong MCHypertension and disrupted blood pressure circadian rhythm in type 2 diabetic db/db mice. Am J Physiol. 2008;295:H1634–41.[Google Scholar]
35. Bagi Z, Erdei N, Toth A, Li W, Hintze TH, Koller A, et al Type 2 diabetic mice have increased arteriolar tone and blood pressure: enhanced release of COX-2-derived constrictor prostaglandins. Arterioscler Thromb Vasc Biol. 2005;25:1610–6.[PubMed][Google Scholar]
36. Habibi J, Aroor AR, Sowers JR, Jia G, Hayden MR, Garro M, et al Sodium glucose transporter 2 (SGLT2) inhibition with empagliflozin improves cardiac diastolic function in a female rodent model of diabetes. Cardiovas Diabetol. 2017;16:9.[Google Scholar]
37. Solini A, Rossi C, Duranti E, Taddei S, Natali A, Virdis A. Saxagliptin prevents vascular remodeling and oxidative stress in db/db mice. Role of endothelial nitric oxide synthase uncoupling and cyclooxygenase. Vas Pharmacol. 2016;76:62–71.[PubMed]
38. Heagerty AM, Heerkens EH, Izzard ASSmall artery structure and function in hypertension. J Cell Mol Med. 2010;14:1037–43.[Google Scholar]
39. Huang A, Yang YM, Feher A, Bagi Z, Kaley G, Sun DExacerbation of endothelial dysfunction during the progression of diabetes: role of oxidative stress. Am J Physiol. 2012;302:R674–81.[Google Scholar]
40. Konior A, Schramm A, Czesnikiewicz-Guzik M, Guzik TJNADPH oxidases in vascular pathology. Antioxid Redox Signal. 2014;20:2794–814.[Google Scholar]
41. Santillo M, Colantuoni A, Mondola P, Guida B, Damiano SNOX signaling in molecular cardiovascular mechanisms involved in the blood pressure homeostasis. Front Physiol. 2015;6:194.[Google Scholar]
42. Kolluru GK, Bir SC, Kevil CGEndothelial dysfunction and diabetes: effects on angiogenesis, vascular remodeling, and wound healing. Int J Vas Med. 2012;2012:918267.[Google Scholar]
43. Maloney E, Sweet IR, Hockenbery DM, Pham M, Rizzo NO, Tateya S, et al Activation of NF-kappaB by palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation. Arterioscler Thromb Vasc Biol. 2009;29:1370–5.[Google Scholar]
44. Aird WCEndothelial cell heterogeneity. Cold Spring Harb Perspect Med. 2012;2:a006429.[Google Scholar]
45. Burridge KA, Friedman MHEnvironment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries. Am J Physiol. 2010;299:H837–46.[Google Scholar]

Circulating metabolites of strawberry mediate reductions in vascular inflammation and endothelial dysfunction in <em>db/db</em> mice<sup><sup><a href="#FN3" rid="FN3" class=" fn">1</a></sup>–<sup><a href="#FN6" rid="FN6" class=" fn">5</a></sup></sup>

Background

Methods

Results

Conclusions

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Pre-clinical model of type 2 diabetes

2.3. Standard diet and strawberry supplemented diet

2.4. Experimental groups

2.5. Measurement of metabolic variables, blood pressure and collection of tissue samples

2.6. Measurement of vascular inflammation

2.7. Measurement of vascular function

2.8. Isolation of ECs from carotid artery and assessment of endothelial effects of strawberry

2.9. Cell culture

2.10. Measuring the effects of serum of strawberry-fed mice on endothelial inflammation

2.11. Data Analysis

2.1. Materials

2.2. Pre-clinical model of type 2 diabetes

2.3. Standard diet and strawberry supplemented diet

2.4. Experimental groups

2.5. Measurement of metabolic variables, blood pressure and collection of tissue samples

2.6. Measurement of vascular inflammation

2.7. Measurement of vascular function

2.8. Isolation of ECs from carotid artery and assessment of endothelial effects of strawberry

2.9. Cell culture

2.10. Measuring the effects of serum of strawberry-fed mice on endothelial inflammation

2.11. Data Analysis

3. Results

3.1. Strawberry supplementation does not alter the systemic metabolic milieu in db/db mice

3.2. Strawberry supplementation reduces vascular inflammation and modestly improves lipid peroxidation in db/db mice

3.3. Blood pressure was lower and endothelial vasorelaxation was improved in the strawberry supplemented db/db mice

3.4. Strawberry supplementation reduces endothelial inflammatory molecules in db/db mice indicating the endothelial effects of strawberry bioactives

3.5. Strawberry supplementation reduces the expression of inhibitor κB kinase (IκKβ) and NOX2 in db/db mice

3.6. Strawberry metabolites suppress endothelial inflammation in MAECs exposed to high glucose and palmitate

3.1. Strawberry supplementation does not alter the systemic metabolic milieu in db/db mice

3.2. Strawberry supplementation reduces vascular inflammation and modestly improves lipid peroxidation in db/db mice

3.3. Blood pressure was lower and endothelial vasorelaxation was improved in the strawberry supplemented db/db mice

3.4. Strawberry supplementation reduces endothelial inflammatory molecules in db/db mice indicating the endothelial effects of strawberry bioactives

3.5. Strawberry supplementation reduces the expression of inhibitor κB kinase (IκKβ) and NOX2 in db/db mice

3.6. Strawberry metabolites suppress endothelial inflammation in MAECs exposed to high glucose and palmitate

4. Discussion

4.1. Limitations

4.2. Conclusions

4.1. Limitations

4.2. Conclusions

Supplementary Material

supplement

supplement

Acknowledgments

Abstract

Background

Methods

Results

Conclusions

Footnotes

References