The authors have declared that no competing interests exist.
Conceived and designed the experiments: DG SS BW. Performed the experiments: DG SS AJ AB. Analyzed the data: DG AJ. Wrote the paper: DG SS AJ AB BW.
Obesity and obesity-associated diseases e.g. cardiovascular diseases and type 2 diabetes are spread worldwide. Anthocyanins are supposed to have health-promoting properties, although convincing evidence is lacking. The aim of the present study was to investigate the effect of anthocyanins on several risk factors for obesity-associated diseases. Therefore, Fischer rats were fed anthocyanin-rich grape-bilberry juice or an anthocyanin-depleted control juice for 10 weeks. Intervention with anthocyanin-rich grape-bilberry juice reduced serum cholesterol and tended to decrease serum triglycerides. No effects were seen for serum non-esterified fatty acids, glucose, and insulin. Anthocyanin-rich grape-bilberry juice intervention reduced serum leptin and resistin, but showed no influence on serum adiponectin and secretion of adipokines from mesenteric adipose tissue. Furthermore, anthocyanin-rich grape-bilberry juice increased the proportion of polyunsaturated fatty acids and decreased the amount of saturated fatty acids in plasma. These results indicate that anthocyanins possess a preventive potential for obesity-associated diseases.
In Western countries with high prevalence of obesity and its associated diseases, cardiovascular diseases and diabetes are severe public health problems. As obesity is spreading worldwide, it is expected that the incidence of these diseases will increase over the coming years
Anthocyanins, a subgroup of flavonoids, occur in red, violet, and blue fruits and vegetables, especially berries. Several health-promoting properties are discussed for anthocyanins, including anti-diabetic properties and beneficial effects on the cardiovascular system
The development of cardiovascular diseases, particularly atherosclerosis, is associated with endothelial dysfunction. Saturated fatty acids (SFA) are known to impair, whereas long-chain n3-polyunsaturated fatty acids (PUFA) improve endothelial function
The adipose tissue secretes several mediators, called adipokines (e.g. adiponectin, leptin, and resistin), which have regulatory functions in the metabolism. Additionally, it has been reported that adipokines are involved in the development of metabolic diseases, such as type 2 diabetes and cardiovascular diseases. High levels of leptin and resistin, occurring in obese individuals, promote the development of insulin resistance, whereas adiponectin seems to prevent insulin resistance
The aim of the present study was to investigate the effect of a 10-week intervention with anthocyanins on several risk factors for diabetes and cardiovascular diseases, as well as on plasma fatty acid composition. One focus of the present study was to mimic the situation in Western countries. Therefore, we used a diet which ensured a Western-style fat intake and focussed on MAT as fat depot. Fischer rats were fed a diet containing 34% energy from fat with high content of SFA, which closely resembles fat intake in Western countries, and received an anthocyanin-rich grape-bilberry juice (ARJ).
This study was approved by the Animal Care Committee of the Regional Administrative Authority Karlsruhe (Permit Number: 35-9185.81/G-221/10) and all animal care and handling were conducted in strict accordance with the guidelines of the German law on animal care.
Male Fischer 344 rats at the age of 10 weeks were purchased from Charles River Laboratories (Sulzfeld, Germany). Rats were housed two animals per cage under temperature- (21±2°C) and humidity-controlled (55–65%) conditions with a 12-h-light/-dark cycle. After one week of adaption animals were randomly assigned to intervention groups (n = 30/group). Blood and organs from six animals per group were used to assess bioavailability of anthocyanins. The anthocyanin group received ARJ, containing 1551 mg anthocyanins/L, the control group received polyphenol-depleted grape-bilberry juice. Pure juices were the only sources of liquids. The major anthocyanins were malvidin-3,5-diglucoside and peonidin-3,5-diglucoside. The composition of the juices and data of anthocyanin analysis has been described in detail elsewhere
For measuring the adipokine release of MAT the method of Paul
For serum preparation blood was collected and the clotted blood was centrifuged at 2000 x g for 10 min. Aliquots of serum were stored at −20°C until further analysis.
For plasma preparation blood was collected in heparinised tubes and centrifuged at 400 x g for 10 min. 0.1 mg BHT/mL plasma was added and aliquots were stored at −20°C until further analysis.
For the quantification of adiponectin (AdipoGen, Liestal, Switzerland), leptin, and resistin (BioVendor, Heidelberg, Germany) in tissue culture supernatant or serum, sandwich ELISAs were performed according to the manufacturer's instructions. For quantification of adipokines in MAT supernatant, the triplicates were pooled and analysed in duplicates.
Glucose (Gluco-Quant, Roche, Mannheim, Germany), insulin (Rat insulin ELISA, Millipore, Schwalbach, Germany), cholesterol (CHOD-PAP, Roche), NEFA (NEFA-HR2, Wako, Neuss, Germany), and triglycerides (GPO-PAP, Roche) were measured using commercial kits according to the manufacturers' instructions.
For analysis of plasma fatty acids a GC method with flame ionization detection was used, which separated FAME ranging from C4 to C26. The detailed method was described earlier
For statistical analysis of effects between the two groups t-tests were performed. If data failed to satisfy variance homogeneity or normal distribution, the Mann-Whitney Rank Sum test was employed. SigmaPlot 11.0 (Systat Software, Erkrath, Germany) was used for all statistical analyses. Differences were considered statistically significant when p < 0.05. All data are presented as mean ± standard deviation.
Data on food, juice, and anthocyanin intake as well as body and organ weights have been presented previously
The intervention with ARJ did not influence serum glucose and insulin levels (
Control | ARJ | p-value | |
Glucose |
1.4±0.1 | 1.4±0.2 | 0.253 |
Insulin |
2.9±1.6 | 3.6±2.5 | 0.464 |
Cholesterol |
1.1±0.1 | 1.0±0.1 | 0.029 |
NEFA |
0.65±0.21 | 0.67±0.17 | 0.614 |
Triglycerides |
3.4±1.0 | 2.9±0.8 | 0.079 |
ARJ: Anthocyanin-rich grape-bilberry juice
The adipokines adiponectin, leptin, and resistin were quantified in serum and MAT culture supernatant. Rats of the ARJ group had lower serum leptin and resistin levels compared to the control group. Adiponectin serum levels did not differ between ARJ and control group (
Concentration of adiponectin, leptin, and resistin in serum of Fischer rats after 10 weeks of intervention with control juice (white bars) or ARJ (grey bars; n = 24/group).
The intervention did not influence the adipokine concentrations in tissue culture supernatants of MAT (
Control | ARJ | p-value | |
Adiponectin | 3.7±0.6 | 3.9±0.6 | 0.21 |
Leptin | 26.9±5.0 | 25.5±5.2 | 0.36 |
Resistin | 13.2±4.0 | 13.1±2.8 | 0.78 |
ARJ: Anthocyanin-rich grape-bilberry juice
The distribution of fatty acids was determined in plasma (
Percentage of SFA, MUFA, and PUFA of total plasma fatty acids of Fischer rats after 10 weeks of intervention with polyphenol-depleted grape-bilberry control juice (white bars) or anthocyanin-rich juice (grey bars; n = 24/group). The smaller bars show the fatty acids with the n-6/n-3-PUFA with the largest increases. FAME: fatty acid methyl ester. * p<0.05
Control | ARJ | p-value | |
C14:0 | 0.70±0.17 | 0.51±0.07 | <0.001 |
C16:0 | 23.74±2.27 | 21.98±1.63 | 0.003 |
C16:1 |
3.12±0.88 | 2.18±0.51 | <0.001 |
C18:0 | 9.08±2.07 | 9.54±1.89 | 0.529 |
C18:1 |
25.98±3.69 | 25.64±3.02 | 0.975 |
C18:1 |
3.01±0.34 | 2.63±0.34 | <0.001 |
C18:2n-6 | 7.30±1.07 | 8.19±0.97 | 0.004 |
C20:4n-6 | 15.78±3.28 | 16.86±2.43 | 0.201 |
C22:4n-6 | 0.57±0.09 | 0.62±0.10 | 0.039 |
C22:5n-6 | 0.76±0.14 | 0.83±0.16 | 0.062 |
C22:5n-3 | 0.45±0,13 | 0.61±0.31 | 0.101 |
C22:6n-3 | 0.95±0.27 | 1.03±0.22 | 0.089 |
n-6/n-3 | 12/1 | 12/1 | 0.990 |
ARJ: Anthocyanin-rich grape-bilberry juice
>0.5% FAME
In the present study, the influence of an ARJ intervention on carbohydrate and lipid metabolism, serum concentrations and MAT secretion of adipokines, and fatty acid profil in plasma was investigated. As we intended to determine the preventive potential of an anthocyanin-rich diet, we decided to use a whole food in form of a juice and not to use supplements for this intervention. Rats of the anthocyanin group ingested approximately 15 mg total anthocyanins per day, which corresponds to 50 mg/kg body weight. As discussed in Graf
For anthocyanins anti-diabetic properties are assumed and human as well as animal studies reported increased insulin sensitivity or lower blood glucose concentrations after an anthocyanin intervention
Dyslipidemia, especially elevated cholesterol and triglyceride levels, is a risk factor for cardiovascular diseases
We further examined the influence of ARJ consumption on fatty acids in plasma. Although the diet was rich in SFA, SFA decreased and PUFA increased in plasma after 10 weeks of ARJ consumption. It has been described by another study that anthocyanin interventions can increase the plasma concentrations of the long chain n-3-PUFA EPA (C20:5n-3) and DHA (C22:6n-3)
The intervention with ARJ resulted in reduced serum concentrations of leptin and resistin, whereas no effect was observed for adiponectin. Data on the influence of anthocyanins on adipokines are rather controversial.
Leptin as well as resistin are elevated in obesity
In contrast to serum concentrations of adipokines, no effect was observed for the secretory activity of MAT regarding adiponectin, leptin, and resistin. Guo
Our results indicate a positive influence of anthocyanins on serum adipokines, which seems to be independent of MAT, the central visceral adipose tissue. Further studies are needed to determine which adipose depots are responsible for the observed changes in serum and under which conditions anthocyanins exert this effect.
In conclusion, a 10 week intervention with ARJ and a diet, which mimicked Western fat intake led to changes in plasma fatty acids as well as to reductions in serum cholesterol, leptin, and resistin concentrations. These results indicate that intake of anthocyanins at physiological doses e.g. via complex foods like juices, might have a preventive potential for obesity-associated diseases such as cardiovascular disease or type 2 diabetes.
We thank J. Geißert, M. Giorgi, S. Merkel, G. Schultheiss, L. Uhlmann, and A. Waldheim for their excellent technical assistance. Prof. Dietrich, Prof. Will and B. Irmler from the Geisenheim Research Centre are thanked for supplying the juices and analysis of the juices.