Consumption of a single serving of red raspberries per day reduces metabolic syndrome parameters in high-fat fed mice

T. Luo , O. Miranda-Garcia , G. Sasaki and N. F. Shay *
College of Agricultural Sciences, Department of Food Science and Technology, Oregon State University, Corvallis, OR, USA. E-mail: neil.shay@oregonstate.edu

Received 12th May 2017 , Accepted 25th September 2017

First published on 28th September 2017


Using an animal model for diet-induced metabolic disease, we have shown previously that the addition of raspberry juice concentrate (RJC) and raspberry puree concentrate (RPC) at a level of 10% of kcal, equivalent to four servings per day, to an obesogenic high-fat, western-style diet (HF) significantly reduced body weight gain, serum resistin levels, and altered the expression of hepatic genes related to lipid metabolism and oxidative stress. This study was designed to examine the effect of a lower level of RJC or RPC consumption, at a level representing a single serving of food per day (2.5% of kcal). For ten weeks, four groups of C57BL/6J mice (n = 8 ea.) were fed: low fat (LF), HF, HF + RJC, or HF + RPC diets. Intake of RJC and RPC decreased final body weight. Hepatic lipid accumulation was significantly decreased in HF + RPC- and HF + RJC-fed mice, compared to HF-fed mice. Further, the relative expression of hepatic genes including Heme oxygenase 1 (Hmox1) and Hormone sensitive lipase (Lipe), were altered by RPC or RJC consumption. In this mouse model of diet-induced metabolic disease, consumption of the equivalent of a single daily serving of either RPC or RJC improved metabolism in mice fed HF diet. We hypothesize that the phytochemicals contained in raspberries, and/or their subsequent metabolites, may be acting to influence gene expression and other regulatory pathways, to produce the metabolic improvements observed in this study.


1. Introduction

Metabolic syndrome is a disorder of energy utilization and storage, which is diagnosed by a co-occurrence of three out of the five following medical conditions: abdominal obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low HDL cholesterol levels.1 Central lipid accumulation is regarded as an obvious symptom for metabolic syndrome.2 Oxidative stress and inflammation are thought to play an important role in the development of metabolic syndrome.3,4

Metabolic syndrome is a concern because it is highly prevalent, also it is linked to several health conditions.5 However, to date, no drugs have been approved for the treatment of metabolic syndrome. Physical activity and dietary modification remain the best strategy to limit metabolic syndrome in the general population.6

Raspberries rank as the third most popular fresh berry in the United States, led only by strawberries and blueberries.7 The United States is the world's third largest producer of raspberries.8 Records of raspberry on health are found as early as medieval times, when wild berries were used for medicinal purposes.9 Although less studied than other berries, a growing body of data suggests important health benefits are associated with red raspberry intake. Daily consumption of red raspberry juice for 12 weeks significantly reduced aortic lipid deposition in hamsters fed an atherogenic diet.10 Five weeks’ feeding of red raspberry extracts at a concentration of 100 or 200 mg kg−1 day−1 demonstrated an antihypertensive effect in spontaneously hypertensive rats.11 In an eight week human study, 300 g fresh berries comprising of 100 g of strawberry puree, 100 g of frozen raspberries, and 100 g of frozen cloudberries per day produced decreased leptin levels, no effect on blood lipids, blood pressure, or platelet function among subjects with symptoms of metabolic syndrome. Bioavailability of berry ellagitannins appears to be dependent on the composition of the gut microbiota.12

Red raspberries are among the few plant foods that provide a source of ellagitannins and anthocyanins. Sanguiin H-6 and lambertianin C are the major ellagitannin identified in red raspberries.13,14 Ellagitannins can be hydrolyzed, spontaneously releasing free ellagic acid. Other phenolic compounds such as ellagic acids, quercetin and kaempferol are also present in red raspberries.9 In the large intestine, ellagic acid and ellagitannins are principally converted to urolithin metabolites. It is considered that urolithin is more bioactive than ellagic acid and ellagitannin: 2–10 μM of urolithin and 0.1–0.4 μM of ellagic acid were found in serum.15 Also, the half-life of urolithins in the human body is longer than that of ellagic acid.15 In addition to their unique polyphenol profile, red raspberries are among the highest whole food sources of dietary fiber, providing 6.5 g fiber per 100 g of fresh weight.16

Previously, using C57BL/6J mice, we demonstrated that the addition of raspberry juice concentrate (RJC, at 10% of kcal) and raspberry puree concentrate (RPC, at 10% of kcal) to an obesogenic western-style diet (HF) containing high levels of saturated fat, cholesterol, and sugar significantly reduced body weight gain and serum resistin, a marker correlating with glucose intolerance and diabetes. Compared to the HF-fed control mice, expression of hepatic genes related to lipid metabolism and oxidative stress were significantly altered in HF + RJC- and HF + RPC-fed mice.17 In the present study, we reduced the content of raspberry in food diet to only 2.5% of daily kcal intake, the equivalent of one serving per day for humans. Our hypothesis is that even the feeding of a single serving of red raspberries per day, as RPC or RJC, will improve the metabolism of obese and diabetic mice fed a HF diet.

2. Materials and methods

2.1 Materials

Raspberry puree concentrate (RPC) and raspberry juice concentrate (RJC) were received as a gift from Milne Fruit Products, Prosser WA.

2.2 C57BL/6J mice and diets

Thirty-two 6-week-old male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and acclimated for two weeks to our facility and the control low fat diet (LF). Semi-purified defined diets were produced by Research Diets, Inc. (New Brunswick, NJ, USA). Mice were then randomly divided into four different groups (n = 8): two control groups were fed either a LF diet containing 10% fat and 70% carbohydrate by energy or a HF group diet containing 45% kcal fat, 20% kcal sucrose, and 1% (w/w) cholesterol. Two other two groups were provided the HF diet containing 2.5% kcal of either RPC (HF + RPC) or RJC (HF + RJC) (Table 1). Diets were macronutrient balanced so the HF control, and two test diets all contained the same amount of carbohydrate, protein, and fat. Mice were kept four per cage in a room maintained at a constant temperature (24 °C), with a 12 h light/dark cycle, and given free access to food and distilled water. During 10 weeks’ feeding, the body weight and food intake of the mice was recorded once a week, with spillage accounted for. All federal, state, and local policies regarding animal use were followed and the animal protocol was approved by the institutional animal care committee (ACUP 4455).
Table 1 Composition of the experimental diets, including LF, HF, HF + RPC, and HF + RJC
Ingredients (g) LF HF HF + RPC HF + RJC
Casein 200 200 197.5 198.5
L-Cysteine 3 3 3 3
Corn starch 506.2 72.8 68.9 68
Maltodextrin 10 125 100 100 100
Sucrose 68.8 172.8 157.8 154
Cellulose, BW200 50 50 48.1 49.4
Soybean oil 25 25 25 25
Lard 20 177.5 176.7 176.3
Mineral mix S10026 10 10 10 10
Dicalcium phosphate 13 13 13 13
Calcium carbonate 5.5 5.5 5.5 5.5
Potassium citrate, 1H2O 16.5 16.5 16.5 16.5
Vitamin mix V10001 10 10 10 10
Choline bitartrate 2 2 2 2
Cholesterol 0 8.5 8.5 8.5
RPC 0 0 94 0
RJC 0 0 0 40
 
kcal%
Protein 20 18 18 18
Carbohydrate 70 36 36 36
Fat 10 46 46 46
kcal g−1 3.8 4.59 4.58 4.50


2.3 Fasting blood glucose and intraperitoneal glucose tolerance test

A glucose tolerance test was performed at week 9. Mice diets were withheld for six hours prior to testing and the test was performed mid-day in the middle of the light cycle. Tail vein blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min using a handheld glucometer (ReliOn, Abbott Laboratories, Abbott Park, IL), after mice were administered glucose (0.1 mg per g b.w.) by intraperitoneal injection. The index of glucose tolerance was indicated as the area under the curve (AUC) using the trapezoidal rule to determine AUC.18,19 The value at t = 0 min was used as a baseline glucose concentration.

2.4 Histological analysis of liver tissue

At week 10, after the mice were sacrificed, liver tissue was collected, fixed in buffered formalin. Tissue was then embedded in paraffin, and three to four 5 μm thick sections were transferred to numbered slides. Slides were then stained with Masson's trichrome stain. Images were acquired using a Nikon Eclipse E400 microscope (Nikon Co., Tokyo, Japan) equipped with an extended digital camera (Q imaging, Surrey, BC, Canada). Lipid droplet percentage, as the ratio of white color area to the total area, was obtained with Adobe Photoshop 7.0, generally following guidelines by Dahab et al.20

2.5 Plasma biomarkers quantitation

Blood samples were collected via cardiac puncture, incubated on ice for 30–60 minutes and centrifuged at 1000g for 15 min at 4 °C, after which serum was collected. Serum insulin level was measured in 96-well plates using ELISA kits (Invitrogen Corporation, Camarillo, CA, USA), following the manufacturer's instructions. Plates were read with a Wallac 1420 Victor2 Microplate Reader (PerkinElmer life and analytical sciences, Turku, Finland).

2.6 Hepatic gene expression

Liver tissue was collected immediately after mice were killed. For each mouse, a 200 mg piece of liver tissue was stored in 200 μL RNAlater® Solution (Life Technologies, Carlsbad, CA, USA) at 4 °C for 24 hours and RNA was isolated from liver homogenates using TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY, USA). Reverse transcription of cDNA used a PrimerScript RT-PCR kit according to the manufacturer's protocol. The expression levels for specific genes were determined by real-time qPCR using SYBR Premix Ex Taq using an ABI 7900HT Real-Time PCR system. Gene expression levels were normalized to the housekeeping gene Rpl30. The real-time qPCR protocol was as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 20 s. The primer sequences used for Ribosomal protein L30 (Rpl30), Heme oxygenase 1 (Hmox1), and Hormone sensitive lipase (Lipe) are:

Rpl30: (F) 5′-GTGGGAGCTCCTTCCTTTCTC-3′, (R) 5′-GACTTTTTCGTCTTCTTTGCGG-3′.

Hmox-1: (F) 5′-CACGCATATACCCGCTACCT-3′, (R) 5′-CCAGAGTGTTCATTAGAGCA-3′.

Lipe: (F) 5′-GACCATCAACCGACCAGGAG-3′, (R) 5′-GGATGGCAGGTGTGAACTGG-3′.

2.7 Statistical analysis

Data are presented as means ± SEM. ANOVA was used to compare sets of data; Tukey's procedure was used for post hoc testing when ANOVA showed significant differences. Significance was established at a level of p < 0.05. All statistical analyses were carried out using GraphPad Prism 6.

3. Results

3.1 Effect of RPC or RJC intake on body weight gain and food intake

Initial body weights in different groups were not significantly different. When C57BL/6J mice were fed the LF control diet, they remained lean and physically normal, however, mice fed the HF control diet had significantly increased final body weights at week ten (29 ± 0.8 g in LF vs. 42 ± 1.2 g in HF). The energy intake in HF control diet-fed mice was also higher than that in LF fed mice.

When compared to fed the HF control diet, RPC and RJC intake significantly reduced final body weight by 15% ± 6%, and 17% ± 3%, respectively, without any significant differences in food intake (Fig. 1).


image file: c7fo00702g-f1.tif
Fig. 1 Body weight and energy intake in C57BL/6J male mice fed a low fat (LF), a high fat (HF) diet alone, HF plus raspberry puree concentrate (HF + RPC) or HF plus raspberry juice concentrate (HF + RJC) during 10 weeks feeding. (A) Body weight of mice fed LF, HF, HF + RPC, or HF + RJC diet at week 10. (B) Energy intake/week/four mouse in LF-, HF-, HF + RPC-, or HF + RJC-fed mice. Bars are mean ± SEM (n = 8). Bars with the same letter are not significantly different from each other, p < 0.05.

3.2 Effect of RPC or RJC consumption on mice tissue weight

As shown in Fig. 2A and B, RPC and RJC consumption has no effect on kidney weight, compared to the kidney weight in HF fed mice. There is a trend that the liver weight and inguinal adipose tissue weight were reduced in HF + RPC and HF + RJC fed mice compared to mice fed the HF control diet, however, no significant difference was observed (p > 0.05).
image file: c7fo00702g-f2.tif
Fig. 2 Organ weight and organ weight/body weight ratios of C57BL/6J male mice fed a low fat (LF), a high fat (HF) diet alone, HF plus raspberry puree concentrate (HF + RPC) or HF plus raspberry juice concentrate (HF + RJC) at week 10. After sacrificing the mice at week 10, the tissue samples including kidney (A, and B), liver (C and D), and inguinal adipose tissue (E and F) were obtained. Bars are mean ± SEM (n = 8). Bars with the same letter are not significantly different from each other, p < 0.05.

3.3 Effect of RPC or RJC consumption on hepatic lipid accumulation

Liver tissue was evaluated for lipid content by histology and image analysis (Fig. 3). More lipid content was observed in the liver of mice fed the HF control diet compared to HF + RPC- and HF + RJC-fed mice.
image file: c7fo00702g-f3.tif
Fig. 3 Hepatic lipid accumulation in C57BL/6J male mice fed a low fat (LF), a high fat (HF) diet alone, HF plus raspberry puree concentrate (HF + RPC) or HF plus raspberry juice concentrate (HF + RJC) at week 10. (A) Representative photomicrographs of liver sections stained with Masson's trichrome for the indicated diet groups, LF, HF, HF + RPC, and HF + RJC. (B) Quantified results of lipid accumulation in LF, HF, HF + RPC, and HF + RJC groups. Bars are mean ± SEM (n = 8); bars with the same letter are not significantly different from each other, p < 0.05. Scale: 500 μm.

Quantified lipid content is shown in Fig. 3B. Compared to mice fed HF control diet, mice fed HF + RPC or HF + RJC had significantly reduced fat content in the liver by 42% ± 5%, and 47% ± 5%, respectively.

3.4 Effects of RPC and RJC on glucose homeostasis

Baseline blood glucose concentration was determined in LF-, HF-, HF + RJC-, and HF + RPC-fed mice. The fasting glucose level was significantly increased in mice fed the HF control diet compared to mice fed the LF control diet. When either RPC or RJC was supplemented to HF diet, fasting glucose levels were decreased to a level that was statistically indistinguishable from the mice fed the LF control diet. However, no influence on AUC was found in HF + RPC- or HF + RJC-fed mice, compared to mice fed the HF control diet (data not shown).

As shown in Fig. 4B, HF + RJC consumption reduced serum insulin concentration compared to mice fed HF control diet. In mice fed HF + RPC, insulin levels were reduced to a level that was statistically indistinguishable from both the LF- and HF-fed mice.


image file: c7fo00702g-f4.tif
Fig. 4 The levels of glucose homeostasis related parameters in serum of C57BL/6J male mice fed a low fat (LF), a high fat (HF) diet alone, HF plus raspberry puree concentrate (HF + RPC) or HF plus raspberry juice concentrate (HF + RJC) at week 10. (A) Baseline blood glucose concentrations. Diets were withheld for six hours, then the glucose concentration was determined from the tail using a handheld glucometer. (B) Plasma insulin concentration. Bars are mean ± SEM (n = 8). Bars with the same letter are not significantly different from each other, p < 0.05.

3.5 Effects of RPC and RJC on hepatic gene expression

Relative hepatic gene expression, including Hmox1 and Lipe was determined in LF-, HF-, HF + RPC-, and HF + RJC-fed mice. RJC supplemented to HF diet dramatically increased both Lipe and Hmox1 (p < 0.05), while intake of RPC produced a significant increase in Lipe and an increase in Hmox that was statistically indistinguishable from both control mice and RJC-fed mice (Fig. 5).
image file: c7fo00702g-f5.tif
Fig. 5 Hepatic gene expression in C57BL/6J male mice fed a low fat (LF), a high fat (HF) diet alone, HF plus raspberry puree concentrate (HF + RPC) or HF plus raspberry juice concentrate (HF + RJC) after 10 weeks feeding. (A) Relative expression of Hmox1. (B) Relative expression of Lipe. Bars are mean ± SEM (n = 8). Bars with the same superscript letter are not significantly different from each other, p < 0.05.

4. Discussion

We have shown previously that the addition of RPC or RJC to a rodent diet at a level equivalent to four food servings per day (10% of daily kcal from raspberry) in a human diet, reduced body weight gain and serum resistin, in a HF diet induced metabolic syndrome in C57BL/6J mice.17

In the present study, the potential of one serving of red raspberries as 2.5% daily kcal of RPC or RJC to remediate symptoms of diet-induced metabolic disease intake was determined. Decreased body weight gain, hepatic lipid accumulation, improved glucose regulation, and alterations in gene expression related to oxidative stress and lipid metabolism were observed in RPC- and RJC-fed mice.

One serving of raspberry contains 50 calories,16 and is a reasonable amount for an individual to consume on a daily or occasional basis. In our animal model, RPC or RJC was added to high fat diets to provide at 2.5% of total dietary energy per day, which is equivalent to a human consuming 50 calories in a typical 2000 calories per day diet. As diet formulations were macronutrient balanced, there are no confounding effects of providing different groups of mice different amounts of protein, fat, or carbohydrate.

Importantly, after 10 weeks of feeding, RPC and RJC intake reduced mice final body weight by 15%, and 17%, respectively, compared to mice fed the HF control diet. In agreement with the body weight gain, the hepatic lipid content was reduced with RPC and RJC consumption (Fig. 3). Although, the baseline blood glucose concentration was not significantly altered in HF + RPC and HF + RJC groups, the insulin level was indeed regulated with RJC consumption (Fig. 4B), showing an ameliorated influence on glucose metabolism. Intake of RPC also showed a change in insulin levels so that after 10 weeks, the insulin levels in HF + RPC-fed mice was not significantly different from LF control mice.

Gene expression was measured for selected genes and demonstrate that the intake of RPC and RJC produced some changes that resulted in gene expressing levels more closely resembling healthy LF-fed control mice vs. the HF-fed control mice. Lipe, catalyzes triglyceride hydrolysis21 and was markedly upregulated in HF + RPC- and HF + RJC-fed mice, consistent with enhanced triglyceride catabolism. This finding is consistent with the decreased hepatic lipid accumulation measured in HF + RPC- and HF + RJC-fed mice (Fig. 3). The other gene reported here, Hmox1, plays a role in protecting against oxidative stress.22 Hepatic Hmox1 gene expression was significantly upregulated in HF + RJC-fed mice, and was at a level intermediate to both control groups and the HF + RJC group. The results suggest that intake of RPC and RJC may protect mice from intracellular oxidative stress.

We hypothesize that the phytochemicals contained in raspberries, and/or their subsequent metabolites, may be acting to produce the metabolic improvements observed in this study. In vitro and in vivo studies have demonstrated that anthocyanins and ellagitannins (via ellagic acid or their urolithin metabolites) could reduce the risk of or reverse metabolically associated pathophysiologies.9

Anthocyanin-rich fraction from red raspberry was shown to suppress inflammatory signaling, such as NF-kB, in LPS-activated RAW264.7 macrophages.23 Decreased measures of inflammation and increased endogenous antioxidant defenses enzymes were reported, when male Wister rats were gavaged with 20 mg kg−1 raspberry ellagitannins for 10 days.24 Urolithin C was reported to reduce TNFα-induced inflammation through the inhibition of histone acetyltransferase (HAT) activity in monocytes.25 Kang et al. also reported that ellagic acid, as well as urolithin A, C, and D, triggered the activation of AMPK pathway, in cultured human adipocytes.26

On the other hand, we hypothesize the fiber also counts for a beneficial compound in red raspberry. As raspberry contains both soluble and insoluble fiber, the contribution of this fruit fiber to the cellulose-based control diets is likely to impact microbiota composition. This area required additional study.

The present study does have some limitations. For example, only the Hmox1 and Lipe mRNA levels was reported, and the expression levels of other genes that are critical to hepatic lipid metabolism and may mediate the raspberries’ action on hepatic fat contents were not examined in this study. Future studies should include this information. Further, information on phytochemical compositions of the raspberry juice concentrate and raspberry puree concentrate are not reported herein, and need further investigation.

5. Conclusion

We found that consumption of the equivalent of a single daily serving of either RPC or RJC for 10 weeks ameliorated the symptoms of metabolic syndrome in C57BL/6J mice fed a high-fat, western-style diet. However, human study is needed to confirm the influence of red raspberries intake on human metabolism.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This research was supported by National Processed Raspberry Council (USA).

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