Lingonberry (Vaccinium vitis-Idaea L) Mobilizes L6 Muscle GLUT4 Transporters and Exerts Anti-Obesity and Antidiabetic Effects in vivo

Research Article

Austin J Endocrinol Diabetes. 2014;1(3): 1012.

Lingonberry (Vaccinium vitis-Idaea L) Mobilizes L6 Muscle GLUT4 Transporters and Exerts Anti-Obesity and Antidiabetic Effects in vivo

Hoda M Eid1,2,5, Antoine Brault1,2, Meriem Ouchfoun1,2, Farah Thong4, Diane Vallerand1,2, Lina Musallam1,2, John T Arnason2,3, Gary Sweeney4 and Pierre S Haddad1,2*

1Natural Health Products and Metabolic Diseases Laboratory, Dept. of Pharmacology, Université de Montrèal, Montreal, Quebec, Canada

2Canadian Institutes of Health Research Team in Aboriginal Antidiabetic Medicines and Montreal Diabetes Research Center, Montreal, Quebec, Canada

3Phytochemistry, Medicinal Plant and Ethnopharmacology Laboratory, Dept. of Biology, University of Ottawa, Ottawa, Ontario, Canada

4Dept. of Biology, York University, Toronto, Ontario, Canada

5Department of Pharmacognosy, University of Beni-seuf, Beni-seuf, Egypt

*Corresponding author: Pierre S. Haddad, Department of Pharmacology, Université de Montréal, P.O. Box 6128, Centre-Ville Station, Montreal, Quebec, H3C 3J7 Canada,

Received: February 10, 2014; Accepted: March 06, 2014; Published: March 13, 2014


Lingonberry (Vaccinium vitis-idaea L.) is an important part of Scandinavian diet. It is also popular in some parts of Europe and North America, and is used to produce confectionary and food products. This plant has been identified among species used by the Cree of EeyouIstchee (northern Quebec) to treat symptoms of diabetes.

In a previous study, the ethanol extract of V. vitis berries enhanced glucose uptake in C2C12 muscle cells through stimulation of AMP–activated protein kinase (AMPK) pathway. In this study, we firstly investigated the effect of this product on the translocation of insulin–sensitive glucose transporters GLUT4 in L6-GLUT4myc skeletal muscle cells. V. vitis extract (200 μg⁄ml, 18h) significantly increased glucose uptake and induced GLUT4 translocation to the cell membrane of L6 cells through an insulin–independent mechanism involving AMPK.

Secondly, we carried out in vivo experiment to validate its antidiabetic effect. The extract was administered to diabetic KKAy mice for 10 days. V. vitis decreased glycaemia, cumulative food intake and body weight. Moreover, V. vitis tended to increase skeletal muscle GLUT4 expression and attenuated hepatic statuses.

These results demonstrate that V. vitis berries represent a promising avenue for the culturally adapted management of obesity and diabetes in Canadian aboriginals.

Keywords: Type 2 diabetes mellitus; Obesity; GLUT4; V. vitis; lingonberry; KKAy mice; AMPK.


Several members of the genus Vaccinium bear edible berries; many of them are reputed for their antidiabetic activity. European blueberry or bilberry (V. myrtillus L.) were widely used in Europe to treat diabetes prior to the discovery of insulin [1]. In one study, unsweetened cranberry juice (V. macrocarpon Ait.) helped to lower blood glucose levels in patients with type 2 diabetes [2]. In addition,Canadian blueberry (V. angustofolium Ait.) has been observed to exhibit antidiabetic activities in cultured skeletal muscle [3]. Finally, a recent study has shown that the bio–transformed blueberry juice incorporated in the drinking water reduced hyperglycemia of diabetic KKAy mice [4].

Aboriginal populations are particularly at risk for developing type 2 diabetes mellitus and its complications. In Canada, the prevalence of diabetes for these populations is at least three times higher than that of the general population and is expected to increase three–fold over the next 20 years [5]. Lingonberry (Vaccinium vitis–idaea L.), known also as mountain cranberry or partridgeberry, is consumed by the Cree communities of EeyouIstchee (CEI, Eastern James Bay region of the Canadian province of Quebec) not only as food but also as medicine to treat symptoms of diabetes including frequent urination [6,7]. Our research team identified his plant during a previous bioactivity screening study [8], as part of a project aiming to provide culturally relevant alternative treatment options for Cree diabetics, whose disease prevalence is among the highest in Canada.

In our previous study, V. vitis was found to increase glucose transport in muscle cells through the activation of AMPK as a response to metabolic stress resulting from a non–toxic disruption of mitochondrial energy transduction [9]. The present study was carried out firstly to determine whether V. vitis increases GLUT4 translocation in skeletal muscle cells as suspected from enhanced glucose transport. We selected L6 myocytes because they express more GLUT4 proteins than our previous C2C12 cellular model and because tools exist to better ascertain GLUT4 translocation to the plasma membrane. Secondly, our objective was to confirm the antidiabetic activity of V. vitis in an in vivo model of type 2 diabetes. KKAy mice are a cross between glucose–intolerant black KK female mice and yellow obese Ay male mice. They are characterized by hyperphagia, insulin resistance, hyperinsulinemia, diabetes, dyslipidemia and hypertension. Therefore, KKAy mice are an excellent model for type 2 diabetes induced by obesity [10]. This model was thus selected to evaluate the in vivo antidiabetic activity of V. vitis.

Materials and Methods

Plant material and extraction

Berries of V. vitis were collected in the Eastern James Bay region, QC, Canada, and kept at −20°C until use. Botanical identity was confirmed by Dr. Alain Currier (Institute de recherché en biologie végétale, Université de Montréal), plant taxonomist on our Team, and voucher specimens were deposited at the Montreal Botanical Garden herbarium (voucher # Whap04–21).

In total 800 g of the berries were freeze–dried (Super Modulo freeze dryer; Thermo Fisher, Ottawa, Ont, Canada) to yield 114 g of dry material. The dry material was then extracted three times for 24 h with ten volumes of 80% ethanol on a mechanical shaker and then filtered under vacuum using Whitman 1 paper. The supernatants were combined and dried using a rotary evaporator (RE 500; Yamato Scientific, Tokyo, Japan) followed by lyophilization.

Cell culture

Rat L6 skeletal muscle cells were grown in minimum essential medium alpha (α–MEM) supplemented with 10% (v⁄v) fetal bovine serum (FBS) in a 5% CO2 at 37°C and used as myoblasts when fully confluent. For differentiation into my tubes, cells were switched to a medium containing 2% FBS for 5–7 days. Cells transfected to stably overexpress GLUT4 harboring mycepitope on the first exofacial loop of the transporter (L6 GLUT4myc cells) were provided by Dr Amira Klip (The Hospital for Sick Children, Toronto, ON, Canada).

Measurement of glucose uptake

L6–GLUT4myccells were cultured in 12–well plates and were used after 5–7 days of differentiation. The cells were serum–starved for 4 h before incubation with the maximum non–toxic concentration of V. vitis (200 μg⁄ml) for 18 h or insulin (100 nM, 20 min). Cells were incubated in transport solution [140 mM NaCl, 20 mM HEPES–Na, 2.5 mM MgSO4, 1 mM CaCl2, 5 mMKCl, 10 μM 2–Deoxy–Glucose and 0.5 μCi⁄ml 2–deoxy–D–[3H] glucose (pH 7.4)] for 5 min at room temperature. Cells were then lysed with 1 M KOH and aliquots were transferred to scintillation vials for 3H radioactivity counting and expressed as fold increase over control. Nonspecific uptake was measured in the presence of cytochalasin B (10 μM) and was subtracted from all values.

Determination of cell surface GLUT4 (OPD assay)

Levels of GLUT4 mycat the cell surface were measured by an antibody–coupled colorimetric assay [11]. Briefly, L6 myoblasts were cultured in 24–well plates until confluence and serum–starved for 4 h before incubation with either V. vitis (200 μg⁄ml) for 18 h or insulin (100 nM) for 20 min. Cells were then quickly washed in ice–cold PBS and incubated with an anti–c–mycantibody (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at 4°C. After that, cells were washed and fixed in 3% para– formaldehyd for 3 min on ice. To neutralize the fixative, cells were incubated with 10 mM glycine in ice–cold PBS for 10 min, and then blocked in 5% goat serum for 30 min. Horseradish peroxides (HRP)–conjugated goat anti–rabbit IgG was then applied for 60 min at 4°C (1:1,000 dilution; Cell Signaling Technologies, Danvers, MA, USA). Cells were washed five times with ice–cold PBS and incubated with O–phenylenediamine dihydrochloride (OPD) reagent (1 ml⁄well) (Sigma–Aldrich, St. Louis,MO, USA) at room temperature for 30 min. To stop the reaction, 0.25 ml of 3 M HCl was added to each well. The supernatant was collected and its absorbance was measured at 492 nm. Absorbance associated with nonspecific binding (primary antibody omitted) was used as a blank.

Animals and in vivo experimental protocols

Study #1: Effect of V. vitis on diabetic KKAy mice. KKAy mice were derived from an in–house colony established using breeding pairs obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice weighing 33–42 g were housed individually and kept for 1 weekon a 12 h light–dark cycle in a temperature controlled chamber and provided with a regular laboratory chow and water ad libitum. The animals were divided into two groups containing seven mice each, as follows: Group 1 diabetic mice, average body weight of 36.7± 0.89,received drinking tap water and served as controls; Group 2 diabetic mice, average body weight of 37.8± 1.21 were administered with 1% V. vitis in drinking water (equivalent to a dose of 4 g⁄ kg of crude berry extract on the first day of treatment and 1.33 g⁄kg thereafter, due to a drop in fluid intake until the end of the experiment). The dose was chosen on the basis of preliminary experiments. In addition, bilberry, another vaccininium species ( ) reputed to possess antidiabetic activities, was administered to rats at a similar dose of 3 g⁄kg⁄day [12]. During the ten days of treatment, body weight as well as food and fluid intake were determined on a daily basis. The non–fasting blood glucose concentration was measured daily using an Accu–Chek glucometer (Roche, Montreal, QC, Canada) by collecting blood from the tip of the tail vein. On the last day of treatment, the mice were anaesthetized, sacrificed and organs such as liver, skeletal muscle, kidney, epididymal fat pad, abdominal fat pad and dorsal fat pad were immediately removed and stored in a −80° C freezer until used. All experimental protocols were approved by the animal experimentation ethics committee of the University of Montreal and carried out in full respect of the guidelines from the Canadian Council for the Care and Protection of Animals.

Study #2: Pair–feeding effect in KKAy diabetic mice

Pair feeding was employed in order to investigate to what extent the blood glucose–lowering effect observed with V. vitis in study #1 could be attributed to the observed reduction in food intake. Animals were allocated into two groups containing seven mice each as follows: Group 1 diabetic mice, weighed 33.74 ± 0.85, were administeredwith 1% V. vitis in drinking water; Group 2 diabetic mice, weighed 33.16 ± 1.55, received drinking water and were pair–fed to group 1 mice. Pair feeding was carried out by measuring the food intake of the ad libitum–fed V. vitis treated mice every 24 h and presenting this amount of food to the pair–fed treated mice with a one–day delay.Food consumption, fluid intake, body weight and blood glucose were recorded daily. At the end of the study, the mice were sacrificed, blood samples were obtained and tissues were harvested as described for study #1.

Study #3: normal C57BL⁄6J mice

To study the effect on V. vitis on blood glucose levels and food intake in normal animals, normal C57BL⁄6J mice were housed as described before and randomly divided into two groups containing seven mice. Group 1 mice (average body weight of 28.8± 0.49) received drinking water and served as control; Group 2 mice (averagebody weight of 28.92 ± 0.41) were administered with 1% V. vitis in drinking water. Both groups were fed regular laboratory chow ad libitum. The experimental protocol lasted for 10 days and was performed as described for study #1.

At the end of each study, the mice were anesthetized using 50 mg⁄kg pentobarbital intraperitoneally and then sacrificed by exsanguinations via the inferior vena cava.

Blood parameters

Plasma insulin and adiponectin levels were determined using radioimmunoassay kits (Linco Research, St–Charles, MO, USA). The levels of serum triglycerides, cholesterol, HDL, LDL, creatinine, alkaline phosphates, AST (Aspirate aminotransferase), ALT (Alanine aminotransferase) and LDH (lactate dehydrogenize) were measured by the Department of Biochemistry of Sainte–Justine’s Children Hospital (Montreal, QC, Canada).

Western blot for proteins involved in glucose and lipid metabolism

The effects of the plant extract on insulin and AMPK signaling pathways in L6 muscle cells were assessed by western immunoblot analysis. Differentiated L6 cells (5–7 day) cultured in 6–well plates were treated with plant extract or vehicle alone (DMSO) for 18 h.Twenty minutes prior to the end of the treatment, insulin (100 nM) or aminoimidazole carboxamide ribonucleotide (AICAR; 1 mM) were added to some vehicle–treated wells as positive controls. For ACC and GLUT4 western blot analysis, samples of muscle of KKAy mice from studies #1 & 2 were used. L6 cells and muscle tissues were homogenized in 1 ml of RIPA lysis buffer (25 mM Tris–HCl pH 7.4, 25 mM NaCl, 0.5 mM EDTA, 1% Triton–X–100, 0.1% SDS) for 30 min on ice and were centrifuged at 12000 x g for 10 min. For all supernatentsamples, a protease inhibitor cocktail was added (Roche, Mannheim, Germany) as well as 1 mM phenylmethanesulfonyl fluoride (PMSF) and phosphates inhibitors (1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM sodium fluoride). Supernatants were then stored at –80oC until analysis. Upon thawing, protein content was assayed by the bicinchoninic acid method standardized to bovine erum albumin (Roche, Laval, QC, Canada) [13]. Lysates were diluted to a concentration of 1 mg⁄ml total protein and boiled for 5 min inreducing sample buffer (62.5 mMTris–HCl pH 6.8, 2% SDS, 10% glycerol, 5% ß–mercaptoethanol and 0.01% bromophenol blue). One hundred μL of each sample were separated on 10% polyacrylamidefull–size gels and transferred to nitro cellulose membrane (Millipore, Bedford, MA, USA). Membranes were blocked for 2 h at room temperature with Tween–20 and 5% skim milk in TBS (20 mM Tris– HCl, pH 7.6 and 137 mM NaCl). Membranes were then incubated overnight at 4°C in blocking buffer with appropriate phospho–specific or pan–specific antibodies against AMPK, ACC, GLUT4 (each at 1: 1000). Membranes were washed 5 times and incubated 1.5 h at roomtemperature in TBS plus Twine 20 with anti–rabbit HRP–conjugated secondary antibodies at 1:50000 to 1:100000 (Jackson Immune research, Cedar lane Laboratories, Horn by, ON, Canada). Revelation was performed using the enhanced chemiluminescence method and blue–light–sensitive film (Amersham Biosciences, Buckinghamshire, England). Gel band intensities were evaluated by densitometry analysis using Image Densitometry software (Version 1.6, National Institutes of Health and Bethesda, MD, USA).

Histological Analysis

Sections of excised livers were placed in the 10% formalin solution and were stained with hematoxylinphloxine saffron (HPS) by the Institute de Recherché en Immunologieet en Cancérologie (IRIC), Department of Histology (Université de Montréal, Montreal, QC, Canada). Each stained liver section was analyzed for the severity of lipid accumulation in the hepatocyte and was then scored based on the percentage of hepatocyte that contained macrovesicular fat: namely, grade 0 (0–5%), grade 1(5–33%), grade 2 (33–66%), and grade3 (66–100%), as previously described [14].

Statistical analysis

In vitro results as well as quantification of western blot data for in vivo studies were analyzed by one–way analysis of variance (ANOVA) using Stat View software (SAS Institute Inc, Cary, NC, USA), with post–hoc analysis as appropriate. Areas under the curve (AUC) were calculated by using PRISM software (Graph Pad, San Diego, CA, USA). Calculations of cumulative changes (body weight, food and liquid intake) were initiated several days prior to the onset of treatment in order to obtain baseline values. For the in vivo studies, Student’s t test for unpaired observations was used. Non–parametric data was analyzed by the Chi square test. Statistical significance was set at p≤0.05. Results are presented as the mean ± SEM for the indicated number of determinations or animals.


V. vitis increases glucose uptake and GLUT4 translocation in L6 my tubes

To confirm that V. vitis increases glucose uptake in L6–GLUT4mycmy tubes as it did in C2C12 cells [9], cells were treated with 200 μg⁄ ml V. vitis for 18 h or 100 nM insulin for 20 min and tested for [3H]– 2–deoxy–D–glucose uptake. V. vitis significantly stimulated glucose uptake by 65 ± 5 % above DMSO, p<0.05. This was comparable to the glucose uptake stimulated by insulin (positive control), whichreached 75 ± 13 % above DMSO (NS as compared to V. vitis; Figure 1a).

Using the OPD assay, we then examined the effect of V. vitis on GLUT4 translocation in L6–GLUT4mycmyoblasts. Our results showed that V. vitis significantly stimulated GLUT4 translocation in these cells with an efficacy very similar to that of insulin (both 1.8 fold increase relative to DMSO; Figure 1b, p<0.05).