Skip to main content Accessibility help
×
Home
Hostname: page-component-55597f9d44-qcsxw Total loading time: 0.716 Render date: 2022-08-10T18:24:35.469Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "useRatesEcommerce": false, "useNewApi": true } hasContentIssue true

Polyphenol-rich blackcurrant extract exerts hypocholesterolaemic and hypoglycaemic effects in mice fed a diet containing high fat and cholesterol

Published online by Cambridge University Press:  22 April 2015

Tyler Benn
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Bohkyung Kim
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Young-Ki Park
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Yue Yang
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Tho X. Pham
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Chai Siah Ku
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Callie Farruggia
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Ellen Harness
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
Joan A. Smyth
Affiliation:
Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, Connecticut06269, USA
Ji-Young Lee*
Affiliation:
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut06269, USA
*
*Corresponding author: J.-Y. Lee, fax +1 860 486 3674, email ji-young.lee@uconn.edu
Rights & Permissions[Opens in a new window]

Abstract

Obesity is associated with an increased risk of metabolic abnormalities, such as hyperlipidaemia and hyperglycaemia. We investigated whether polyphenol-rich blackcurrant extract (BCE) can prevent high fat/high cholesterol (HF/HC) diet-induced metabolic disturbances in mice. Male C57BL/6J mice were fed a modified AIN-93M diet containing HF/HC (16 % fat, 0·25 % cholesterol, w/w) or the same diet supplemented with 0·1 % BCE (w/w) for 12 weeks. There were no differences in total body weight and liver weight between groups. Plasma total cholesterol (TC) and glucose levels were significantly lower in BCE group than in controls, while plasma TAG levels were not significantly different. There was a decreasing trend in hepatic TAG levels, and histological evaluation of steatosis grade was markedly lower in the livers of mice fed BCE. Although the mRNA levels of major regulators of hepatic cholesterol metabolism, i.e. 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) and LDL receptor (LDLR), were not significantly altered by BCE supplementation, protein expression of mature sterol-regulatory element-binding protein and LDLR was significantly increased with no change in HMGR protein. The expression of proprotein convertase subtilisin/kexin type 9 that facilitates LDLR protein degradation, as well as one of its transcriptional regulators, i.e. hepatocyte nuclear factor 4α, was significantly decreased in the livers of mice fed BCE. Taken together, BCE supplementation decreased plasma TC and glucose, and inhibited liver steatosis, suggesting that this berry may be consumed to prevent metabolic dysfunctions induced by diets high in fat and cholesterol.

Type
Full Papers
Copyright
Copyright © The Authors 2015 

Obesity is among several global health concerns and a leading preventable cause of death, second only to tobacco smoking and elevated blood pressure( Reference Danaei, Ding and Mozaffarian 1 ). Existing research has identified that a subpopulation of obese subjects appears to be metabolically healthy and less likely to progress to states of obesity-associated disease( Reference Wildman, Muntner and Reynolds 2 ). Therefore, it is not obesity alone that has resulted in health risk but rather the progression of obesity to metabolic dysfunction, including hyperlipidaemia, hyperglycaemia and insulin resistance( Reference Bluher 3 ). These abnormal metabolic conditions can lead to the development of CVD, type 2 diabetes and non-alcoholic fatty liver disease. Therefore, effective dietary strategies to prevent obesity-associated metabolic diseases are needed.

Epidemiological studies have shown that consumption of fruits and vegetables is negatively correlated with the risk of chronic diseases( Reference Ford and Mokdad 4 , Reference Joshipura, Hu and Manson 5 ). Berries are of particular interest due to their suggested health benefits, which are largely attributed to their high contents of polyphenols, anthocyanins in particular( Reference Rossi, Serraino and Dugo 6 Reference Fernandes, Faria and Azevedo 11 ). While blueberry, cranberry, blackberry and raspberry are commonly consumed in the USA, blackcurrant (Ribes nigrum) has only recently gained popularity. Blackcurrant is a rich source of anthocyanins and vitamin C( Reference Tabart, Kevers and Evers 12 ). We previously reported that blackcurrant contains four major anthocyanins, including delphinidin-3-rutinoside, cyanidin-3-rutinoside, delphinidin-3-glucoside and cyanidin-3-glucoside, contributing to approximately 98 % of total anthocyanins in blackcurrant( Reference Lee, Kim and Yang 13 ). Blackcurrant is known to have higher antioxidant capacity than other commonly consumed berries due to its high polyphenol contents( Reference Kulling and Rawel 14 Reference Wu, Gu and Prior 16 ).

Studies have shown that blackcurrant exerts anti-inflammatory, antioxidant and anti-microbial effects, which provide potential health benefits against hypertension, CVD, neurodegenerative disease, ocular diseases and hypercholesterolaemia( Reference Tabart, Kevers and Evers 12 , Reference Gopalan, Reuben and Ahmed 17 Reference Skoczynska, Jedrychowska and Poreba 25 ). In addition, blackcurrant consumption has been shown to improve insulin sensitivity and inhibit inflammation( Reference Gopalan, Reuben and Ahmed 17 ). A study using mice fed a high fat (HF) diet showed that blackcurrant lowered body weight, body fat, plasma glucose, insulin, alanine transaminase (ALT), inflammatory markers and liver TAG( Reference Heyman, Axling and Blanco 26 ). A clinical study with healthy women also showed that blackcurrant improved postprandial metabolic responses to sucrose, i.e. a slower rise in serum glucose and insulin, and improved glycaemic profile( Reference Torronen, Kolehmainen and Sarkkinen 27 ). However, mechanisms of action for the health benefits of blackcurrant have been limitedly understood. In the present study, we sought to investigate potential roles and mechanisms of polyphenol-rich blackcurrant extract (BCE) in the prevention of obesity-associated metabolic abnormalities in mice fed a diet high in fat and cholesterol.

Materials and methods

Animal care and diet

Male C57BL/6J mice (Jackson Laboratory) at 15 weeks of age were randomly assigned to a control (n 11) or a BCE group (n 13). After 1 week of acclimation, the control group of mice was fed a modified AIN-93 diet( Reference Reeves, Nielsen and Fahey 28 , Reference Reeves 29 ) containing HF/high cholesterol (HC) (15 % fat, 0·25 % cholesterol, w/w), while the BCE group was on the HF/HC diet supplemented with 0·1 % of BCE (w/w). The standardised BCE powder containing 25 % anthocyanins and 40 % polyphenols was provided by Artemis International, Inc. Based on body surface normalisation to a 70 kg individual( Reference Reagan-Shaw, Nihal and Ahmad 30 ), 0·1 % BCE containing 25 % anthocyanins is equivalent to daily consumption of approximately 540 mg BCE and 135 mg anthocyanins in human subjects. As the average daily intake of anthocyanins per person has been estimated to be approximately 200 mg in the USA( Reference Kuhnau 31 ), we believe the dietary level of berry extracts is attainable in human subjects. Mice were housed in a controlled environment with 12-h light–12-h dark cycles and were fed ad libitum throughout the study. Body weight and food consumption were recorded weekly, and blood draws were performed monthly from the lateral tail vein. After 12 weeks on the experimental diets, mice were fasted for 8 h and anaesthetised by injecting ketamine/xylazine (100/10 mpk) (Henry Schein Animal Health). Blood samples were collected into a 2-ml BD vacutainer containing EDTA by cardiac puncture and mice were killed by exsanguination followed by cervical dislocation. Blood was centrifuged at 1500  g for 10 min at 4°C. Livers were weighed, and subsamples were snap frozen in liquid N2 and stored at − 80°C until use or fixed in 10 % formalin. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Connecticut.

Liver histology and hepatic lipid content

Formalin-fixed livers were processed to paraffin, sections were cut at 4–5 μm and stained with haematoxylin and eosin at the Connecticut Veterinary Medical Diagnostic Laboratory, Department of Pathobiology and Veterinary Science, University of Connecticut (Storrs, CT). Histological evaluation was performed twice to ensure consistency by a veterinary pathologist (J. A. S.) who was blinded to the study treatments. All sections were graded for steatosis at a × 200 magnification. A commonly used semi-quantitative scoring system was used to assess the severity of hepatic steatosis as described previously( Reference Bruno, Dugan and Smyth 32 ). For steatosis, grades 0, I, II and III indicate 0, < 33, 33–64 and ≥ 65 % of hepatocytes are steatotic, respectively. Grade III was later sub-divided into III-1, III-2 and III-3 to represent mice with 65–70, 71–89 ≥ 90 % steatotic hepatocytes within the hepatic parenchyma. The ranking was made within grading bands based on the mean score of all × 200 fields.

Plasma chemistry and liver lipids

Plasma concentrations of total cholesterol (TC) and TAG were determined by enzymatic analysis using a cholesterol reagent from Pointe Scientific and an L-Type TG-M kit from Wako Chemical USA, respectively, as we described previously( Reference Kim, Ku and Pham 21 ). Plasma ALT and glucose levels were determined using a Liquid ALT (SGPT) Reagent Set and a Liquid Glucose (Oxidase) Reagent Set from Pointe Scientific according to the manufacturer's protocol. Lipids were extracted from liver samples by Folch's method( Reference Folch, Lees and Sloane Stanley 33 ), and TC and TAG were determined by enzymatic analysis as described earlier.

Gene expression analysis by quantitative real-time PCR

Total RNA was extracted from liver samples using TRIzol reagent (Invitrogen). Quantitative RT-PCR analysis was conducted to measure the expression of genes related to fat, cholesterol and glucose metabolism using the SYBR Green procedure and CFX96 real-time PCR detection system (Bio-Rad) as described previously( Reference Kim, Ku and Pham 34 Reference Yang, Seo and Nguyen 40 ). Primer sequences were designed according to GenBank database using the Beacon Designer software (Premier Biosoft) and the sequences will be available upon request. Ribosomal protein large P0 was used as an internal control.

Western blot analysis

Liver lysates were prepared and Western blot analysis was performed as described previously( Reference Rasmussen, Blobaum and Park 41 ). The following antibodies were used: LDL receptor (LDLR; Abcam), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR; Santa Cruz Biotechnology), mature sterol-regulatory element binding protein 2 (mSREBP-2; Abcam) and β-actin (Sigma). The blots were developed using horseradish peroxidase (Thermo Fisher Scientific) and densitometry analysis was performed using Chemidoc XRS+ (Bio-Rad) and Image Lab software (Bio-Rad). β-Actin was used as a loading control.

Statistical analysis

Unpaired Student's t tests were conducted to compare the mean difference between groups using GraphPad InStat 6 (GraphPad Software, Inc.). An α-level of P< 0·05 was considered statistically significant and all data are expressed as means with their standard errors.

Results

Effect of blackcurrant extract supplementation on body weight and plasma chemistry

After 12 weeks on the HF/HC diets, there were no significant differences in final body weight and body weight change between control and BCE-fed mice (Table 1). Diet consumption (data not shown) and plasma ALT levels were not significantly altered by BCE supplementation. While plasma TAG was not significantly different between groups, plasma TC and glucose levels were significantly less in mice fed BCE than in control mice (Fig. 1 and Table 1).

Table 1 Body weight, plasma chemistry and liver lipid levels of C57BL/6J mice fed a high fat/high cholesterol diet supplemented with 0·1 % blackcurrant extract (BCE) (w/w) for 12 weeks (Mean values with their standard errors)

ALT, alanine aminotransferase; TC, total cholesterol.

* Mean value was significantly different from that of control (P< 0·05).

Fig. 1 Plasma lipid levels of male C57BL/6J mice fed a high fat/high cholesterol control or 0·1 % (w/w) blackcurrant extract (BCE) supplemented diet for 12 weeks. (a) Plasma total cholesterol (TC), n 11 for control and n 12 for BCE. (b) Plasma TAG, n 11 for control and n 13 for BCE. Values are means, with their standard errors represented by vertical bars. * Mean value was significantly different from that of control (P< 0·05). To convert cholesterol in mg/dl to mmol/l, multiply by 0·0259. To convert TAG in mg/dl to mmol/l, multiply by 0·0113.

Decreased liver steatosis in mice fed a blackcurrant extract-supplemented diet

Control and BCE-fed mice had no significant differences in liver weight and TC contents (Table 1). However, there was a decreasing trend of liver TAG in the mice fed BCE (P= 0·072). Furthermore, histological examination of liver samples demonstrated that there was a marked reduction in lipid accumulation in five (38·5 %) of the BCE mice (steatosis score grade 1), while the control mice all had steatosis grade 3 (Fig. 2). Furthermore, on sub-categorising grade 3 mice, most (72 %) of the control mice had grade III-3 hepatic steatosis, whereas approximately only 37·5 % of grade 3 BCE-fed mice had grade III-3 (Table 2).

Fig. 2 Histological sections of haematoxylin and eosin-stained liver of male C57BL/6J mice after 12 weeks on a high fat/high cholesterol (HF/HC) control or 0·1 % (w/w) blackcurrant extract (BCE) supplemented diet. Liver section of a mouse fed an HF/HC control diet (steatosis grade 3) (a), and that from a mouse fed a BCE-supplemented diet (steatosis grade 1) (b). Scale bar = 100 μm.

Table 2 Histological analysis for steatosis grade in the livers of C57BL/6J mice fed a high fat/high cholesterol control diet or diet supplemented with 0·1 % blackcurrant extract (BCE) (w/w)

Reduced expression of lipogenic genes in hepatic tissue by blackcurrant extract supplementation

To gain mechanistic insights into the TC-lowering effect of BCE, we measured the hepatic expression of LDLR and HMGR, and sterol-regulatory element binding protein 2 (SREBP-2), a transcriptional regulator of LDLR and HMGR. mRNA abundance of LDLR and HMGR did not differ between the control and BCE groups (Fig. 3(a)). However, protein levels of mSREBP-2 and LDLR were significantly higher in BCE-fed mice than in controls, with no difference in HMGR protein levels (Fig. 3(b) and (c)). The expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), an enzyme known for LDLR protein degradation, and its transcriptional factor, i.e. hepatocyte nuclear factor (HNF)4α, was also significantly repressed by BCE supplementation (Table 3). The expression of two lipogenic genes, SREBP-1c and fatty acid synthase (FAS), was lower in the livers of BCE-fed mice than that of controls, but FAS protein was not decreased by BCE supplementation (data not shown). mRNA levels of genes involved in fatty acid oxidation, such as carnitine palmitoyltransferase 1 (CPT-1)α, CPT-1β and acylCoA oxidase 1, were not significantly altered by BCE supplementation. There was no significant difference in the hepatic expression of gluconeogenic genes, such as phosphenolpyruvate carboxykinase and glucose-6-phosphatase, between the two groups.

Fig. 3 Expression of mRNA and protein levels of lipogenic genes in the livers of male C57BL/6J mice fed a high fat/high cholesterol control or 0·1 % (w/w) blackcurrant extract (BCE) supplemented diet for 12 weeks. (a) mRNA expression. (b) Protein levels (quantification). (c) Western blot image. Values are means, with their standard errors represented by vertical bars (n 11 for control () and n 13 for BCE ()). LDLR, LDL receptor; HMGR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; mSREBP-2, mature sterol-regulatory element binding protein 2. *Mean value was significantly different from that of control (P< 0·05).

Table 3 mRNA expression of genes in the livers of C57BL/6J mice fed a high fat/high cholesterol control diet or diet supplemented with 0·1 % blackcurrant extract (BCE) (w/w)* (Mean values with their standard errors, n 11 for control and n 13 for BCE)

SREBP1c, sterol-regulatory element binding protein 1c; FAS, fatty acid synthase; SCD-1, stearyol CoA desaturase 1; CPT-1, carnitine palmitoyltransferase 1; ACOX-1, acyl CoA oxidase 1; PEPCK, phosphenolpyruvate carboxykinase; G6P, glucose-6-phosphatase; PCSK9, proprotein convertase subtilisin/kexin type 9; HNF, hepatocyte nuclear factor.

* Values are relative expression to control.

Discussion

With the increasing obesity epidemic, co-morbidities of obesity, notably insulin resistance, type 2 diabetes, CVD and non-alcoholic fatty liver disease, are major health problems in the USA( Reference Ruiz-Nunez, Pruimboom and Dijck-Brouwer 42 ). Dyslipidaemia and hyperglycaemia commonly associated with obesity are likely to contribute to the pathogenesis of metabolic diseases. Therefore, identification of dietary products effective in lowering blood lipids and blood glucose would be beneficial for reducing disease risk. Blackcurrant farming has a short history in the USA, but the berry has garnered significant attention due to its potential health benefits. We previously showed that blackcurrant anthocyanins exert potent antioxidant and anti-inflammatory effects( Reference Lee, Kim and Yang 13 ). In the present study, we found that BCE supplementation lowered plasma TC, which is likely attributable, at least in part, to the inhibition of PCSK9-dependent LDLR protein degradation in the liver. Furthermore, BCE also decreased fat accumulation in the liver as well as plasma glucose without altering the expression of genes involved in lipogenesis, fatty acid oxidation or gluconeogenesis. Therefore, the inhibition of hepatic steatosis and hyperglycaemia by BCE is presumed to be secondary to the effects of BCE on extra-hepatic tissues, such as skeletal muscle.

Induction of LDLR expression and activity in the liver is one of the preventive/therapeutic goals to lower circulating cholesterol. Statins, the most prescribed cholesterol-lowering drugs, inhibit HMGR activity and therefore increase LDLR expression( Reference Grundy, Cleeman and Merz 43 ). The induction of LDLR expression primarily depends on SREBP-2, a well-known transcriptional regulator of LDLR, which also up-regulates HMGR expression( Reference Horton, Goldstein and Brown 44 , Reference Sato 45 ). When cellular cholesterol levels are high, insulin-induced genes bind to SREBP-2 in complex with SREBP cleavage-activating protein (SCAP) in the endoplasmic reticulum, preventing the translocation of the SREBP-2/SCAP complex to the Golgi( Reference Ikonen 46 ). Upon depletion of cellular cholesterol, SREBP-2/SCAP is released from insulin-induced genes and transported to the Golgi, where the complex undergoes two-step proteolytic cleavage to release N-terminal transcriptional activation domain, i.e. mSREBP-2, which induces LDLR and HMGR transcription. In the present study, despite a significant increase in mSREBP-2 protein in the livers of BCE-fed mice, LDLR and HMGR mRNA levels were not significantly altered. Hepatic LDLR protein levels, however, were increased by approximately 80 % in the BCE group compared with controls. The result suggests that BCE is likely to increase LDLR protein at post-transcriptional levels.

Recent studies have suggested that PCSK9 plays an important role in the post-transcriptional regulation of LDLR expression. PCSK9 is highly expressed in the liver and intestine, and it promotes LDLR protein degradation by lysosomes( Reference Li, Tumanut and Gavigan 47 Reference Park, Moon and Horton 49 ). Gain-of-function mutations in PCSK9 lead to familial hypercholesterolaemia( Reference Seidah, Benjannet and Wickham 50 , Reference Abifadel, Varret and Rabes 51 ), while loss-of-function mutations are present in hypocholesterolaemic subjects( Reference Cohen, Pertsemlidis and Kotowski 52 ). Therefore, the inactivation of PCSK9 has emerged as a therapeutic target to lower LDL cholesterol concentrations. We found that there was an approximately 40 % decrease in hepatic PCSK9 expression in the BCE group compared with controls, suggesting that decreased PCSK9 expression may be responsible, at least in part, for increased hepatic LDLR protein in BCE-fed mice. Several transcription factors have been suggested to regulate PCSK9 expression. Studies have shown that both LDLR and PCSK9 are activated by SREBP when cellular cholesterol is depleted( Reference Dubuc, Chamberland and Wassef 53 , Reference Maxwell, Soccio and Duncan 54 ). Costet et al. ( Reference Costet, Cariou and Lambert 55 ) also demonstrated that PCSK9 expression is induced by insulin via SREBP-1c. However, in the present study, we did not observe a change in LDLR mRNA, while PCSK9 was decreased in the livers of the BCE-fed group. In contrast, there was a decreasing trend for SREBP-1c mRNA expression in the BCE group (P= 0·052). Given that LDLR expression is under the regulation of SREBP-2 rather than SREBP-1c( Reference Brown and Goldstein 56 ), it can be presumed that reduced SREBP-1c expression by BCE may contribute to the decrease in hepatic PCSK9 expression. HNF1α is also known to transcriptionally regulate PCSK9 expression in cooperation with mSREBP-2( Reference Li, Dong and Park 57 ). Despite increased mSREBP-2 protein, PCSK9 mRNA was repressed in the livers of BCE-fed mice. Therefore, the present results do not support the role of HNF1α/SREBP-2 in mediating the effect of BCE on LDLR expression. Interestingly, BCE supplementation significantly decreased HNF4α expression by approximately 40 %, whereas HNF1α expression was not significantly altered. Ai et al. ( Reference Ai, Chen and Han 58 ) suggested that repression of HNF4α and HNF1α can decrease PCSK9 expression, increasing hepatic LDLR protein levels. At present, it is not clear how HNF and SREBP play a role in the hepatic regulation of PCSK9 by BCE and further investigation is necessary to gain better mechanistic insight.

Histological analysis demonstrated that liver steatosis was reduced in mice fed BCE compared to controls. To determine mechanisms of action, the expression of genes involved in lipogenesis and fatty acid oxidation was measured. Although BCE supplementation significantly decreased FAS mRNA in the liver, its protein levels were not different between groups (data not shown). Furthermore, genes related to mitochondrial fatty acid oxidation, i.e. CPT-1α and CPT-1β, showed a trend towards a decrease, but not an increase, in the livers of mice fed BCE. The mRNA expression of acylCoA oxidase 1, an important enzyme for peroxisomal fatty acid oxidation, was not altered by BCE supplementation. Therefore, the inhibitory action of BCE in the development of liver steatosis is not likely attributed to lipogenesis or fatty acid oxidation. Of interest is our recent report that in the skeletal muscle of mice fed BCE, the expression of genes related to energy expenditure and mitochondrial biogenesis, including PPARα, PPARδ, uncoupling protein (UCP)-2, UCP-3 and mitochondrial transcription factor A, were significantly increased( Reference Benn, Kim and Park 59 ). Furthermore, we did not detect any significant changes in the expression of genes for lipid metabolism in the adipose tissue. These observations support that the decrease in liver steatosis may be secondary to the effect of BCE on energy metabolism in the skeletal muscle. It should also be noted that BCE supplementation significantly decreased plasma fasting glucose levels by approximately 35 %. The hepatic expression of gluconeogenic genes, i.e. glucose-6-phosphatase and phosphenolpyruvate carboxykinase, was not significantly altered by BCE. UCP-2 and UCP-3 play a critical role in glucose and lipid metabolism( Reference Diano and Horvath 60 , Reference Jia, Zhang and Ge 61 ), and over-expression of Ucp-3 in skeletal muscle displayed lower fasting plasma glucose and insulin( Reference Clapham, Arch and Chapman 62 ). Therefore, we propose the beneficial effects of BCE supplementation in the prevention of liver steatosis and hyperglycaemia are likely attributed to enhanced energy utilisation in the skeletal muscle. Future study should be warranted to test this possibility.

In conclusion, the present study demonstrated that dietary supplementation of BCE rich in polyphenols reduced the percentage mice with severe steatosis, hypercholesterolaemia, hyperglycaemia and liver steatosis in mice fed an HF and HC diet. Repressive effect of BCE on the hepatic expression of PCSK9, resulting in increased LDLR protein, is presumed to be responsible for the cholesterol-lowering effect of BCE. To our knowledge, this is the first study that demonstrates the modulation of PCSK9/LDLR axis by dietary components for lowering plasma TC levels. Another important finding of the present study is the reduction of fasting glucose and liver steatosis by BCE supplementation, which we attribute to enhanced energy utilisation in the skeletal muscle. Although detailed molecular mechanisms of action for the health-promoting effects of BCE and identification of bioactive compounds that exert the effects need further investigation, the present study strongly suggests that BCE may be consumed to prevent various metabolic dysfunctions related to HF and HC diet.

Acknowledgements

This work was supported by USDA Hatch CONS00872 and Multi-state Hatch CONS00916 to J.-Y. L.

T. B. played a major role in conducting experiments and contributed to manuscript preparation; B. K., Y.-K. P., Y. Y., T. X. P., C. F. and E. H. contributed to experiments; J. A. S. performed histological evaluation and reviewed the manuscript; J.-Y. L. designed the experiments and contributed to data analysis and manuscript preparation.

All authors claim no conflicts of interest.

References

1 Danaei, G, Ding, EL, Mozaffarian, D, et al. (2009) The preventable causes of death in the United States: comparative risk assessment of dietary, lifestyle, and metabolic risk factors. PLoS Med 6, e1000058.CrossRefGoogle ScholarPubMed
2 Wildman, RP, Muntner, P, Reynolds, K, et al. (2008) The obese without cardiometabolic risk factor clustering and the normal weight with cardiometabolic risk factor clustering: prevalence and correlates of 2 phenotypes among the US population (NHANES 1999–2004). Arch Intern Med 168, 16171624.CrossRefGoogle Scholar
3 Bluher, M (2013) Adipose tissue dysfunction contributes to obesity related metabolic diseases. Best Pract Res Clin Endocrinol Metab 27, 163177.CrossRefGoogle ScholarPubMed
4 Ford, ES & Mokdad, AH (2001) Fruit and vegetable consumption and diabetes mellitus incidence among U.S. adults. Prev Med 32, 3339.CrossRefGoogle ScholarPubMed
5 Joshipura, KJ, Hu, FB, Manson, JE, et al. (2001) The effect of fruit and vegetable intake on risk for coronary heart disease. Ann Intern Med 134, 11061114.CrossRefGoogle ScholarPubMed
6 Rossi, A, Serraino, I, Dugo, P, et al. (2003) Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radic Res 37, 891900.CrossRefGoogle Scholar
7 Zhu, Y, Ling, W, Guo, H, et al. (2013) Anti-inflammatory effect of purified dietary anthocyanin in adults with hypercholesterolemia: a randomized controlled trial. Nutr Metab Cardiovasc Dis 23, 843849.CrossRefGoogle ScholarPubMed
8 Boivin, D, Blanchette, M, Barrette, S, et al. (2007) Inhibition of cancer cell proliferation and suppression of TNF-induced activation of NF-κB by edible berry juice. Anticancer Res 27, 937948.Google ScholarPubMed
9 Devareddy, L, Hooshmand, S, Collins, JK, et al. (2008) Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis. J Nutr Biochem 19, 694699.CrossRefGoogle ScholarPubMed
10 Wang, LS & Stoner, GD (2008) Anthocyanins and their role in cancer prevention. Cancer Lett 269, 281290.CrossRefGoogle ScholarPubMed
11 Fernandes, I, Faria, A, Azevedo, J, et al. (2010) Influence of anthocyanins, derivative pigments and other catechol and pyrogallol-type phenolics on breast cancer cell proliferation. J Agric Food Chem 58, 37853792.CrossRefGoogle ScholarPubMed
12 Tabart, J, Kevers, C, Evers, D, et al. (2011) Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum . J Agric Food Chem 59, 47634770.CrossRefGoogle ScholarPubMed
13 Lee, SG, Kim, B, Yang, Y, et al. (2014) Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism. J Nutr Biochem 25, 404411.CrossRefGoogle ScholarPubMed
14 Kulling, SE & Rawel, HM (2008) Chokeberry (Aronia melanocarpa) – a review on the characteristic components and potential health effects. Planta Med 74, 16251634.CrossRefGoogle ScholarPubMed
15 Li, W, Hydamaka, A, Lowry, L, et al. (2009) Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn. Cent Eur J Biol 4, 499506.Google Scholar
16 Wu, X, Gu, L, Prior, RL, et al. (2004) Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. J Agric Food Chem 52, 78467856.CrossRefGoogle Scholar
17 Gopalan, A, Reuben, SC, Ahmed, S, et al. (2012) The health benefits of blackcurrants. Food Funct 3, 795809.CrossRefGoogle ScholarPubMed
18 Huebbe, P, Giller, K, de Pascual-Teresa, S, et al. (2012) Effects of blackcurrant-based juice on atherosclerosis-related biomarkers in cultured macrophages and in human subjects after consumption of a high-energy meal. Br J Nutr 108, 234244.CrossRefGoogle ScholarPubMed
19 Jurgoński, A, Juśkiewicz, J, Zduńczyk, Z, et al. (2014) Polyphenol-rich extract from blackcurrant pomace attenuates the intestinal tract and serum lipid changes induced by a high-fat diet in rabbits. Eur J Nutr 53, 16031613.CrossRefGoogle ScholarPubMed
20 Finne Nielsen, IL, Elbol Rasmussen, S, Mortensen, A, et al. (2005) Anthocyanins increase low-density lipoprotein and plasma cholesterol and do not reduce atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits. Mol Nutr Food Res 49, 301308.CrossRefGoogle Scholar
21 Kim, B, Ku, CS, Pham, TX, et al. (2013) Aronia melanocarpa (chokeberry) polyphenol-rich extract improves antioxidant function and reduces total plasma cholesterol in apolipoprotein E knockout mice. Nutr Res 33, 406413.CrossRefGoogle ScholarPubMed
22 Valcheva-Kuzmanova, S, Kuzmanov, K, Mihova, V, et al. (2007) Antihyperlipidemic effect of Aronia melanocarpa fruit juice in rats fed a high-cholesterol diet. Plant Foods Hum Nutr 62, 1924.CrossRefGoogle ScholarPubMed
23 Valcheva-Kuzmanova, S, Kuzmanov, K, Tancheva, S, et al. (2007) Hypoglycemic and hypolipidemic effects of Aronia melanocarpa fruit juice in streptozotocin-induced diabetic rats. Methods Find Exp Clin Pharmacol 29, 101105.CrossRefGoogle ScholarPubMed
24 Jurgonski, A, Juskiewicz, J & Zdunczyk, Z (2008) Ingestion of black chokeberry fruit extract leads to intestinal and systemic changes in a rat model of prediabetes and hyperlipidemia. Plant Foods Hum Nutr 63, 176182.CrossRefGoogle Scholar
25 Skoczynska, A, Jedrychowska, I, Poreba, R, et al. (2007) Influence of chokeberry juice on arterial blood pressure and lipid parameters in men with mild hypercholesterolemia. Pharmacol Rep 2007, 177182.Google Scholar
26 Heyman, L, Axling, U, Blanco, N, et al. (2014) Evaluation of beneficial metabolic effects of berries in high-fat fed C57BL/6J mice. J Nutr Metab 2014, 403041.CrossRefGoogle ScholarPubMed
27 Torronen, R, Kolehmainen, M, Sarkkinen, E, et al. (2012) Postprandial glucose, insulin, and free fatty acid responses to sucrose consumed with blackcurrants and lingonberries in healthy women. Am J Clin Nutr 96, 527533.CrossRefGoogle ScholarPubMed
28 Reeves, PG, Nielsen, FH, Fahey, GC, et al. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123, 19391951.Google Scholar
29 Reeves, PG (1997) Components of the AIN-93 diets as improvements in the AIN-76A diet. J Nutr 127, 838S841S.Google ScholarPubMed
30 Reagan-Shaw, S, Nihal, M & Ahmad, N (2008) Dose translation from animal to human studies revisited. FASEB J 22, 659661.CrossRefGoogle ScholarPubMed
31 Kuhnau, J (1976) The flavonoids. A class of semi-essential food components: their role in human nutrition. World Rev Nutr Diet 24, 117191.CrossRefGoogle ScholarPubMed
32 Bruno, RS, Dugan, CE, Smyth, JA, et al. (2008) Green tea extract protects leptin-deficient, spontaneously obese mice from hepatic steatosis and injury. J Nutr 138, 323331.Google ScholarPubMed
33 Folch, J, Lees, M & Sloane Stanley, GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226, 497509.Google ScholarPubMed
34 Kim, B, Ku, CS, Pham, TX, et al. (2012) Aronia melanocarpa (chokeberry) polyphenol rich extract improves antioxidant function and reduces total plasma cholesterol in apolipoprotein E knockout mice. Nutr Res 33, 406413.CrossRefGoogle ScholarPubMed
35 Kim, B, Park, Y, Wegner, CJ, et al. (2013) Polyphenol-rich black chokeberry (Aronia melanocarpa) extract regulates the expression of genes critical for intestinal cholesterol flux in Caco-2 cells. J Nutr Biochem 24, 15641570.CrossRefGoogle ScholarPubMed
36 Ku, CS, Pham, TX, Park, Y, et al. (2013) Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes. Biochim Biophys Acta 1830, 29812988.CrossRefGoogle ScholarPubMed
37 Ku, CS, Rasmussen, HE, Park, Y, et al. (2011) Unsaturated fatty acids repress the expression of ATP-binding cassette transporter A1 in HepG2 and FHs 74 Int cells. Nutr Res 31, 278285.CrossRefGoogle ScholarPubMed
38 Park, Y, Pham, TX & Lee, J (2012) Lipopolysaccharide represses the expression of ATP-binding cassette transporter G1 and scavenger receptor class B, type I in murine macrophages. Inflamm Res 61, 465472.CrossRefGoogle Scholar
39 Yang, Y, Park, Y, Cassada, DA, et al. (2011) In vitro and in vivo safety assessment of edible blue-green algae, Nostoc commune var. sphaeroides Kützing and Spirulina plantensis . Food Chem Toxicol 49, 15601564.CrossRefGoogle ScholarPubMed
40 Yang, Y, Seo, JM, Nguyen, A, et al. (2011) Astaxanthin-rich extract from the green alga Haematococcus pluvialis lowers plasma lipid concentrations and enhances antioxidant defense in apolipoprotein E knockout mice. J Nutr 141, 16111617.CrossRefGoogle ScholarPubMed
41 Rasmussen, HE, Blobaum, KR, Park, YK, et al. (2008) Lipid extract of Nostoc commune var. sphaeroides Kutzing, a blue-green alga, inhibits the activation of sterol regulatory element binding proteins in HepG2 cells. J Nutr 138, 476481.Google ScholarPubMed
42 Ruiz-Nunez, B, Pruimboom, L, Dijck-Brouwer, DA, et al. (2013) Lifestyle and nutritional imbalances associated with Western diseases: causes and consequences of chronic systemic low-grade inflammation in an evolutionary context. J Nutr Biochem 24, 11831201.CrossRefGoogle Scholar
43 Grundy, SM, Cleeman, JI, Merz, CN, et al. (2004) Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 110, 227239.CrossRefGoogle ScholarPubMed
44 Horton, JD, Goldstein, JL & Brown, MS (2002) SREBPs: activator of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109, 11251131.CrossRefGoogle Scholar
45 Sato, R (2010) Sterol metabolism and SREBP activation. Arch Biochem Biophys 501, 177181.CrossRefGoogle ScholarPubMed
46 Ikonen, E (2008) Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 9, 125138.CrossRefGoogle ScholarPubMed
47 Li, J, Tumanut, C, Gavigan, JA, et al. (2007) Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity. Biochem J 406, 203207.CrossRefGoogle ScholarPubMed
48 McNutt, MC, Lagace, TA & Horton, JD (2007) Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells. J Biol Chem 282, 2079920803.CrossRefGoogle Scholar
49 Park, SW, Moon, YA & Horton, JD (2004) Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J Biol Chem 279, 5063050638.CrossRefGoogle ScholarPubMed
50 Seidah, NG, Benjannet, S, Wickham, L, et al. (2003) The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc Natl Acad Sci U S A 100, 928933.CrossRefGoogle ScholarPubMed
51 Abifadel, M, Varret, M, Rabes, JP, et al. (2003) Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 34, 154156.CrossRefGoogle ScholarPubMed
52 Cohen, J, Pertsemlidis, A, Kotowski, IK, et al. (2005) Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat Genet 37, 161165.CrossRefGoogle ScholarPubMed
53 Dubuc, G, Chamberland, A, Wassef, H, et al. (2004) Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 24, 14541459.CrossRefGoogle ScholarPubMed
54 Maxwell, KN, Soccio, RE, Duncan, EM, et al. (2003) Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice. J Lipid Res 44, 21092119.CrossRefGoogle ScholarPubMed
55 Costet, P, Cariou, B, Lambert, G, et al. (2006) Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c. J Biol Chem 281, 62116218.CrossRefGoogle ScholarPubMed
56 Brown, MS & Goldstein, JL (2009) Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL. J Lipid Res 50, S15S27.CrossRefGoogle ScholarPubMed
57 Li, H, Dong, B, Park, SW, et al. (2009) Hepatocyte nuclear factor 1α plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine. J Biol Chem 284, 2888528895.CrossRefGoogle Scholar
58 Ai, D, Chen, C, Han, S, et al. (2012) Regulation of hepatic LDL receptors by mTORC1 and PCSK9 in mice. J Clin Invest 122, 12621270.CrossRefGoogle ScholarPubMed
59 Benn, T, Kim, B, Park, Y-K, et al. (2014) Polyphenol-rich blackcurrant extract prevents inflammation in diet-induced obese mice. J Nutr Biochem 25, 10191025.CrossRefGoogle ScholarPubMed
60 Diano, S & Horvath, TL (2012) Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism. Trends Mol Med 18, 5258.CrossRefGoogle Scholar
61 Jia, JJ, Zhang, X, Ge, CR, et al. (2009) The polymorphisms of UCP2 and UCP3 genes associated with fat metabolism, obesity and diabetes. Obes Rev 10, 519526.CrossRefGoogle ScholarPubMed
62 Clapham, JC, Arch, JR, Chapman, H, et al. (2000) Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean. Nature 406, 415418.CrossRefGoogle ScholarPubMed
Figure 0

Table 1 Body weight, plasma chemistry and liver lipid levels of C57BL/6J mice fed a high fat/high cholesterol diet supplemented with 0·1 % blackcurrant extract (BCE) (w/w) for 12 weeks (Mean values with their standard errors)

Figure 1

Fig. 1 Plasma lipid levels of male C57BL/6J mice fed a high fat/high cholesterol control or 0·1 % (w/w) blackcurrant extract (BCE) supplemented diet for 12 weeks. (a) Plasma total cholesterol (TC), n 11 for control and n 12 for BCE. (b) Plasma TAG, n 11 for control and n 13 for BCE. Values are means, with their standard errors represented by vertical bars. * Mean value was significantly different from that of control (P< 0·05). To convert cholesterol in mg/dl to mmol/l, multiply by 0·0259. To convert TAG in mg/dl to mmol/l, multiply by 0·0113.

Figure 2

Fig. 2 Histological sections of haematoxylin and eosin-stained liver of male C57BL/6J mice after 12 weeks on a high fat/high cholesterol (HF/HC) control or 0·1 % (w/w) blackcurrant extract (BCE) supplemented diet. Liver section of a mouse fed an HF/HC control diet (steatosis grade 3) (a), and that from a mouse fed a BCE-supplemented diet (steatosis grade 1) (b). Scale bar = 100 μm.

Figure 3

Table 2 Histological analysis for steatosis grade in the livers of C57BL/6J mice fed a high fat/high cholesterol control diet or diet supplemented with 0·1 % blackcurrant extract (BCE) (w/w)

Figure 4

Fig. 3 Expression of mRNA and protein levels of lipogenic genes in the livers of male C57BL/6J mice fed a high fat/high cholesterol control or 0·1 % (w/w) blackcurrant extract (BCE) supplemented diet for 12 weeks. (a) mRNA expression. (b) Protein levels (quantification). (c) Western blot image. Values are means, with their standard errors represented by vertical bars (n 11 for control () and n 13 for BCE ()). LDLR, LDL receptor; HMGR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; mSREBP-2, mature sterol-regulatory element binding protein 2. *Mean value was significantly different from that of control (P< 0·05).

Figure 5

Table 3 mRNA expression of genes in the livers of C57BL/6J mice fed a high fat/high cholesterol control diet or diet supplemented with 0·1 % blackcurrant extract (BCE) (w/w)* (Mean values with their standard errors, n 11 for control and n 13 for BCE)

You have Access
25
Cited by

Save article to Kindle

To save this article to your Kindle, first ensure coreplatform@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about saving to your Kindle.

Note you can select to save to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be saved to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

Polyphenol-rich blackcurrant extract exerts hypocholesterolaemic and hypoglycaemic effects in mice fed a diet containing high fat and cholesterol
Available formats
×

Save article to Dropbox

To save this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Dropbox account. Find out more about saving content to Dropbox.

Polyphenol-rich blackcurrant extract exerts hypocholesterolaemic and hypoglycaemic effects in mice fed a diet containing high fat and cholesterol
Available formats
×

Save article to Google Drive

To save this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Google Drive account. Find out more about saving content to Google Drive.

Polyphenol-rich blackcurrant extract exerts hypocholesterolaemic and hypoglycaemic effects in mice fed a diet containing high fat and cholesterol
Available formats
×
×

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *